Genes and gene combinations for enhanced corn performance

ABSTRACT

The present invention identifies a number of transcription factors of corn, genes encoding the transcription factors, and methods to enhance characteristics of corn such as higher photosynthesis rates, higher photosynthetic electron transport rates, reduced photorespiration rates, higher biomass yield or content, higher seed yield, improved harvest index, higher oil content, improved nutritional composition, improved nitrogen use efficiency, drought resistance, flood resistance, disease resistance, salt tolerance, higher C02 assimilation rate, and lower transpiration rate in a plant by upregulating the genes encoding the transcription factors. Compositions of the invention comprise polypeptide sequences, polynucleotide sequences, variants, orthologs, and fragments thereof. Methods comprise introducing into corn plants systems that increase the expression or activity of transcription factors of the invention. Methods and compositions also provide corn plants with enhanced performance.

FIELD OF THE INVENTION

The present invention relates generally to corn transcription factorgene targets, genetic engineering technologies, genome editing materialsand methods for upregulating the expression of those gene targets aloneor in combinations and more particularly, to corn plants havingincreased expression of those gene targets such that they have improvedperformance in soil as compared to the same plant having normalexpression of those genes.

BACKGROUND OF THE INVENTION

The world faces a major challenge in the next 35 years to meet theincreased demands for food production to feed a growing globalpopulation, which is expected to reach 9 billion by the year 2050. Foodoutput will need to be increased by up to 60% in view of the growingpopulation.

Maize which is also known as corn together with wheat, rice and soybeanprovides nearly two thirds of global agricultural calories (Citation:Ray D K, Mueller N D, West P C, Foley J A (2013) Yield Trends AreInsufficient to Double Global Crop Production by 2050. PLoS ONE8(6):e66428. doi:10.1371/journal.pone.0066428). In the United Statesalone in 2017 around 90 million acres of corn was planted with an annualharvest of around 1.6 billion bushels making it the most valuable foodand feed crop. Corn seed genetically engineered for pest resistanceand/or herbicide tolerance is the dominant value driver in the US seedsector. Increasing the field performance of corn and in particular grainyield is critical to addressing global food security and is a majorobjective of the global seed companies.

Since the beginning of genome sequencing, researchers have testedthousands of plant genes individually in corn using genetic engineeringtechniques to increase or decrease the level of activity of the targetgene product. However, other than large numbers of patent applications,including a significant number of theoretical patent applications in theUnited States fisting tens of thousands of genes in patent Claims (forexample, US 2005/0108791; US 2009/0158452; US 2011/0258734; US2013/0074202; and US 2012/0017292), the vast majority of which are basedpurely on DNA sequence homology and with no experimental data, there hasbeen really no significant technical breakthrough or commercialdevelopments using this approach. The long lists of potential cropimprovement benefits, together with the very long lists of potentialgenes for achieving such benefits, is illustrative of just how little isactually taught or reduced to practice regarding specific gene targetsto improve crop performance in these applications and is analogous topointing to a dictionary and indicating there is a great work ofliterature contained in it. In reality, and absent data to the contrary,probably greater than 99% of the gene sequences listed in these broadcases will have either no meaningful impact or possibly be detrimentalto performance. Therefore the need to identify specific corntranscription factor genes for upregulation to significantly improve theperformance of corn remains an unmet need.

In the late 1980's and early 1990's, genetic engineering of transgenicplants was used for the first time to develop crops which are herbicidetolerant, or pest or disease resistant, by introducing genes from themost readily available source at the time, microorganisms, to impartthese new functionalities. Unfortunately, “transgenic plants” or “GMOcrops” or “biotech traits” are not widely accepted in a number ofdifferent jurisdictions and are subject to a regulatory approval processwhich is very time consuming and prohibitively expensive. The currentregulatory framework for transgenic plants results in significant costs(˜$136 million per trait; McDougall, P. 2011, The cost and time involvedin the discovery, development, and authorization of a new plantbiotechnology derived trait. Crop Life International, website:croplife.org/wp-content/uploads/pdffiles/Getting-a-Biotech-Crop-to-Market-Phillips-McDougall-Study.pdf) andlengthy product development timelines that limit the number oftechnologies that are brought to market. These risks have severelyimpaired private investment and the adoption of innovation in thiscrucial sector. Recent changes in the regulations governing geneticallymodified crops by USDA-APHIS in the United States and new technologiessuch as genome editing have begun to change this situation. For example,a corn plant which has been genetically engineered to modify theactivity of a corn gene using only DNA sequences from corn, technicallydescribed as cis-genic not transgenic, may be classified asnon-regulated provided the engineered corn plant contains no foreign DNAsequences. Advances in genome editing technologies provide anopportunity to precisely remove or insert DNA sequences in the plantgenome of interest to inactivate specific plant genes or to alter theirexpression by modifying their promoter sequences to improve plantperformance (Belhaj, K. 2013, Plant Methods, 9, 39; Khandagale & Nadal,2016, Plant Biotechnol Rep, 10, 327). Genetically engineered plants madeusing this approach contain no foreign DNA sequences and may also becategorized as non-regulated by USDA-APHIS. In both cases however, theregulatory status of the engineered plants are appropriately subject tothe usual criteria for approval of any new plant variety.

Clearly there is a need in corn to identify specific transcriptionfactors whose expression can be modified using only corn DNA sequencesalone or in combinations to improve corn crop performance.

BRIEF SUMMARY OF THE INVENTION

It is an objective of this invention to provide specific transcriptionfactor genes for corn as well as the methods, DNA and RNA sequences formodifying or editing these transcription factor genes to increase theirexpression or activity and improve the performance of corn plants. It isa further objective of this invention to provide corn plants, which havebeen modified according to this invention and which have improvedperformance characteristics in the field as compared to the same cornbefore it was modified as disclosed herein.

Accordingly, provided herein is a method for modifying a corn plant, themethod comprising upregulating, in the corn plant, one or morepolynucleotides or polypeptides selected from among the following:

(a) one or more polypeptides comprising SEQ ID NOS: 87, 88, or 89;

(b) one or more polypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16,18, 20, 24, 26, 28, 30, 32, 41, 43 or 48;

(c) one or more of the polypeptides set forth in (a) having at least85%, 90%, 95% or higher sequence identity to one or more of thepolypeptides set forth in (b);

(d) one or more polynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13,15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47;

(e) one or more polynucleotides having at least 85%, 90%, 95% or highersequence identity to one or more of the polynucleotides set forth in(d); or

(f) one or more polypeptides encoded by one or more of thepolynucleotides set forth in (d) or (e).

In accordance with the method, the one or more upregulated polypeptidescan be transcription factors. Also in accordance with the method, theone or more upregulated polynucleotides can encode transcriptionfactors.

In various aspects of the method, the one or more upregulatedpolynucleotides or polypeptides exhibit at least a change in expressionor at least a two-fold change in expression as compared to that of acontrol plant.

In certain aspects, the expression of the transcription factor gene isupregulated using traditional genetic engineering techniques such thatone or more additional copies of the transcription factor gene isinserted into the corn genome under the transcriptional control ofpromoters which are heterologous to the transcription factor gene. Suchrecombinant or chimeric gene constructs are well known in the art.Preferably the method of introducing the additional copy of thetranscription factor gene does not introduce any non-corn or foreign DNAsequences, or where any foreign DNA sequences used during the process ofconstructing the modified corn plant are subsequently removed.

In certain aspects, the expression of the transcription factor gene isaccomplished by deletion, insertion and/or substitution of one or morenucleotides to increase gene expression using gene editing techniquesusing a CRISPR nuclease selected from Cas nuclease, Cas9 nuclease, CasXnuclease, CasY nuclease, a Cpf1 nuclease, a C2c1 nuclease, a C2c2nuclease (Cas13a nuclease), NgAgo nuclease, or a C2c3 nuclease. Forinstance, one or more polynucleotide sequences can be upregulated bytargeting a guide polynucleotide to a target site selected from apromoter, a promoter element, a terminator or a coding sequence of thepolynucleotide sequence using a CRISPR/Cas system to form a complexsuitable for editing a corn genome. Alternatively, transcriptionactivator-like effector nucleases (TALENs) or zinc finger nuclease (ZFN)techniques can be used for editing instead of a CRISPR nuclease.

The methods can be used to produce modified corn plants exhibiting oneor more enhanced characteristics selected from higher photosynthesisrates, higher photosynthetic electron transport rates, highernon-photochemical quenching, reduced photorespiration rates, higherbiomass yield or content, higher seed yield, improved harvest index,higher seed oil content, improved nutritional composition, improvednitrogen use efficiency, drought resistance, flood resistance, diseaseresistance, salt tolerance, higher CO₂ assimilation rate, or lowertranspiration rate

Also provided herein is a modified corn plant comprising:

(a) one or more polypeptides comprising SEQ ID NOS: 87, 88, or 89;

(b) one or more polypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16,18, 20, 24, 26, 28, 30, 32, 41, 43 or 48;

(c) one or more of the polypeptides set forth in (a) having at least85%, 90%, 95% or higher sequence identity to one or more of thepolypeptides set forth in (b);

(d) one or more polynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13,15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47;

(e) one or more polynucleotides having at least 85%, 90%, 95% or highersequence identity to one or more of the polynucleotides set forth in(d); or

(f) one or more polypeptides encoded by one or more of thepolynucleotides set forth in (d);

wherein the one or more polypeptides of (a), (b), (c), or (f) or the oneor more polynucleotides of (d) or (e) are upregulated.

In accordance with the modified corn plant, the one or more upregulatedpolypeptides can be transcription factors. Also in accordance with themodified corn plant, the one or more upregulated polynucleotides canencode transcription factors.

In various aspects of the modified corn plant, the one or moreupregulated polynucleotides or polypeptides exhibit at least a change inexpression or at least a two-fold change in expression as compared tothat of a control plant.

Again, in some embodiments the expression of the transcription factorgene is upregulated using traditional genetic engineering techniquessuch that one or more additional copies of the gene is inserted into thecorn genome under the transcriptional control of corn promoters whichare heterologous to the transcription factor gene. Such recombinant orchimeric gene constructs are well known in the art. Preferably themethod of introducing the additional copy of the transcription factorgene does not introduce any non-corn or foreign DNA sequences or whereany foreign DNA sequences are subsequently removed.

In some embodiments, the polynucleotide sequence encoding one or moretranscription factors are upregulated by DNA insertion, deletion,insertion and/or substitution of one or more nucleotides, site-specificmutagenesis, chemical mutagenesis, using gene editing techniques such asCRISPR nuclease selected from Cas nuclease, Cas9 nuclease, CasXnuclease, CasY nuclease, a Cpf1 nuclease, a C2c1 nuclease, a C2c2nuclease (Cas13a nuclease), a C2c3 nuclease, or a NgAgo nuclease, or byusing TALEN or ZFN techniques. For instance, one or more polynucleotidesequence can be upregulated by targeting a guide polynucleotide to atarget site selected from a promoter, a terminator or a coding sequenceof the polynucleotide sequence using a CRISPR/Cas system to form acomplex suitable for editing a plant.

Compositions useful for overexpression of a transcription factor usingtransgenic or cis-genic technologies described herein are alsodisclosed.

The compositions include a recombinant nucleic acid molecule comprising:

(a) one or more polynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13,15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47;

(b) one or more polynucleotides having at least 85%, 90%, 95% or highersequence identity to one or more of the polynucleotides set forth in(a); or

(c) a fragment of any one of the polynucleotides set forth in (a) or (b)that regulates gene expression; and

further comprising a polynucleotide heterologous to the one or morepolynucleotides of (a) or (b) or the one or more fragments of (c).

The compositions also include a recombinant polypeptide moleculecomprising:

(a) one or more polypeptides comprising SEQ ID NOS: 87, 88, or 89;

(b) one or more polypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16,18, 20, 24, 26, 28, 30, 32, 41, 43 or 48;

(c) one or more of the polypeptides set forth in (a) having at least85%, 90%, 95% or higher sequence identity to one or more of thepolypeptides set forth in (b); or

(d) one or more fragments of any one of the polypeptides set forth in(a), (b), or (c) that regulates gene expression; and

further comprising a polypeptide heterologous to the one or morepolypeptides of (a), (b), or (c) or the one or more fragments of (d).

The compositions also include a recombinant nucleic acid moleculecomprising: (a) a promoter sequence functional in corn, operably linkedto (b) a polynucleotide selected from SEQ ID NOS: 3, 5, 7, 9, 13, 15,17, 19, 23, 25, 27, 29, 31, 40, 42 or 47 encoding a transcription factorgene operably linked to (c) a terminator sequence functional in corn.

The compositions also include the following:

(a) one or more polynucleotides encoding one or more of SEQ ID NOS:52-55 or one or more of SEQ IDS NOS: 75-86 that regulate the expressionof the transcription factor genes encoded by SEQ ID NOS: 3, 5, 7, 9, 13,15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47 in corn; or

(b) a DNA construct targeting the one or more polynucleotides encodingone or more of SEQ ID NOS: 52-55 or one or more of SEQ IDS NOS: 75-86that comprises:

-   -   (i) an expression cassette for a polynucleotide sequence        encoding a CRISPR nuclease containing a promoter sequence        functional in corn; operably linked to a CRISPR nuclease with        codon usage appropriate for use in corn that is flanked by        nuclear localization sequences (NLS) to ensure delivery of the        enzyme into the nuclei; and flanked at the 3′ end by a        terminator sequence functional in corn;    -   (ii) one or more expression cassettes for one or more sgRNAs to        direct the CRISPR nuclease to the appropriate desired nuclease        cut site(s), each cassette containing: a promoter sequence        functional in corn that is appropriate for the expression of        sgRNAs (i.e. plant, and preferably monocot, RNA polymerase III        promoters, such as U6 and U3); DNA encoding an RNA guide        sequence of ˜20 nucleotides; DNA encoding a guide RNA scaffold        (gRNA Sc) which when combined with the previously described RNA        guide sequence forms a functional sgRNA; and a poly        T-termination signal;    -   (iii) a promoter replacement cassette to be inserted in the        double stranded break created by the Cas nuclease at the sgRNA        target sequence(s) containing: a DNA fragment homologous to the        genomic DNA region flanking the 5′ double stranded break site;        the promoter to be inserted; and a DNA fragment homologous to        the genomic DNA region flanking the 3′ double stranded break        site; where the homologous regions direct the insertion of the        new promoter fragment by the plant's endogenous repair        mechanisms; or    -   (iv) an expression cassette for a selectable marker containing:        a promoter sequence functional in corn, operably linked to a        selectable marker appropriate for corn, flanked by a poly        T-termination signal.

Such DNA constructs can provide for enhanced characteristics selectedfrom higher photosynthesis rates, higher photosynthetic electrontransport rates, higher non-photochemical quenching, reducedphotorespiration rates, higher biomass yield or content, higher seedyield, improved harvest index, higher seed oil content, improvednutritional composition, improved nitrogen use efficiency, droughtresistance, flood resistance, disease resistance, salt tolerance, higherCO₂ assimilation rate, or lower transpiration rate.

Exemplary embodiments include the following.

Embodiment 1: A method for modifying a corn plant, the method comprisingupregulating, in the corn plant, one or more polynucleotides orpolypeptides selected from among the following:

(a) one or more polypeptides comprising SEQ ID NOS: 87, 88, or 89;

(b) one or more polypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16,18, 20, 24, 26, 28, 30, 32, 41, 43 or 48;

(c) one or more of the polypeptides set forth in (a) having at least85%, 90%, 95% or higher sequence identity to one or more of thepolypeptides set forth in (b);

(d) one or more polynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13,15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47;

(e) one or more polynucleotides having at least 85%, 90%, 95% or highersequence identity to one or more of the polynucleotides set forth in(d); or

(f) one or more polypeptides encoded by one or more of thepolynucleotides set forth in (d) or (e).

Embodiment 2: The method of embodiment 1, further comprising growing themodified plant under conditions whereby the modified plant exhibits oneor more enhanced characteristics as compared to a control plant grownunder similar conditions.

Embodiment 3: The method of embodiment 1 or embodiment 2, wherein theone or more upregulated polynucleotides comprise SEQ ID NOS: 3, 5, 7, 9,13, 15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47.

Embodiment 4: The method of any one of embodiments 1-3, wherein the oneor more upregulated polynucleotides or polypeptides exhibit at least achange in expression or at least a two-fold change in expression ascompared to that of a control plant.

Embodiment 5: The method of embodiment 4, wherein the change inexpression is accomplished by introducing a transgene for one or moreglobal transcription factors, wherein the transgene comprises apolynucleotide selected from SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23,25, 27, 29, 31, 40, 42 or 47.

Embodiment 6: The method of any one of embodiments 1-5, wherein the oneor more upregulated polynucleotides or polypeptides are upregulated byinsertion and/or substitution of one or more nucleotides, site-specificmutagenesis, chemical mutagenesis, targeting induced local lesions ingenomes (TILLING), gene editing techniques using CRISPR nucleaseselected from Cas nuclease, Cas9 nuclease, CasX nuclease, CasY nuclease,a Cpf1 nuclease, a C2c1 nuclease, a C2c2 nuclease (Cas13a nuclease), ora C2c3 nuclease, NgAgo nuclease, TALEN or ZFN techniques.

Embodiment 7: The method of embodiment 6, wherein the one or moreupregulated polynucleotides or polypeptides are upregulated by targetingone or more guide polynucleotides to one or more target sites selectedfrom a promoter, a terminator, or a coding sequence of the one or morepolynucleotides set forth in (d) or (e).

Embodiment 8: The method of any one of embodiments 1-7, wherein themodified plant exhibits one or more enhanced characteristics selectedfrom higher photosynthesis rates, higher photosynthetic electrontransport rates, higher non-photochemical quenching, reducedphotorespiration rates, higher biomass yield or content, higher seedyield, improved harvest index, higher seed oil content, improvednutritional composition, improved nitrogen use efficiency, droughtresistance, flood resistance, disease resistance, salt tolerance, higherCO₂ assimilation rate, or lower transpiration rate.

Embodiment 9: The method of embodiment 8, wherein the modified plantexhibits an increase in seed oil content or seed yield as compared to acontrol plant.

Embodiment 10: The method of embodiment 9, wherein the seed oil contentof the modified plant is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% or higher relative to the control plant.

Embodiment 11: The method of any one of embodiments 8-10, wherein themodified plant exhibits an increase in photosynthetic electron transportrate as compared to a control plant.

Embodiment 12: The method of embodiment 11, wherein the photosyntheticelectron transport rate of the modified plant is increased by 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or higher relative to thecontrol plant.

Embodiment 13: A modified corn plant comprising:

(a) one or more polypeptides comprising SEQ ID NOS: 87, 88, or 89;

(b) one or more polypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16,18, 20, 24, 26, 28, 30, 32, 41, 43 or 48;

(c) one or more of the polypeptides set forth in (a) having at least85%, 90%, 95% or higher sequence identity to one or more of thepolypeptides set forth in (b);

(d) one or more polynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13,15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47;

(e) one or more polynucleotides having at least 85%, 90%, 95% or highersequence identity to one or more of the polynucleotides set forth in(d); or

(f) one or more polypeptides encoded by one or more of thepolynucleotides set forth in (d);

wherein the one or more polypeptides of (a), (b), (c), or (f) or the oneor more polynucleotides of (d) or (e) are upregulated.

Embodiment 14: The modified plant of embodiment 13, wherein the modifiedplant exhibits one or more enhanced characteristics as compared to acontrol plant grown under similar conditions.

Embodiment 15: The modified plant of embodiment 13 or embodiment 14,wherein the one or more upregulated polynucleotides or polypeptidesexhibit at least a change in expression or at least a two-fold change inexpression as compared to that of a control plant.

Embodiment 16: The modified plant of embodiment 15, wherein the changein expression is accomplished by introducing a transgene for one or moreglobal transcription factors, wherein the transgene comprises apolynucleotide selected from SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23,25, 27, 29, 31, 40, 42 or 47.

Embodiment 17: The modified plant of any one of embodiments 13-16,wherein the one or more upregulated polynucleotides or polypeptides areupregulated by insertion and/or substitution of one or more nucleotides,site-specific mutagenesis, chemical mutagenesis, targeting induced locallesions in genomes (TILLING), gene editing techniques using CRISPRnuclease selected from Cas nuclease, Cas9 nuclease, CasX nuclease, CasYnuclease, a Cpf1 nuclease, a C2c1 nuclease, a C2c2 nuclease (Cas13anuclease), or a C2c3 nuclease, NgAgo nuclease, TALEN or ZFN techniques

Embodiment 18: The modified plant of embodiment 17, wherein the one ormore upregulated polynucleotides or polypeptides are upregulated bytargeting one or more guide polynucleotides to one or more target sitesselected from a promoter, a terminator, or a coding sequence of the oneor more polynucleotides set forth in (d) or (e).

Embodiment 19: The modified plant of any one of embodiments 13-18,wherein the modified plant comprises one or more enhancedcharacteristics selected from higher photosynthesis rates, higherphotosynthetic electron transport rate, higher non-photochemicalquenching, reduced photorespiration rates, higher biomass yield orcontent, higher seed yield, improved harvest index, higher seed oilcontent, improved nutritional composition, improved nitrogen useefficiency, drought resistance, flood resistance, disease resistance,salt tolerance, higher CO₂ assimilation rate, or lower transpirationrate.

Embodiment 20: The modified plant of embodiment 19, wherein the modifiedplant exhibits an increase in seed oil content or seed yield as comparedto a control plant.

Embodiment 21: The modified plant of embodiment 20, wherein the seed oilcontent of the modified plant is increased by 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100% or higher relative to the control plant.

Embodiment 22: The modified plant of any one of embodiments 19-21,wherein the modified plant exhibits an increase in photosyntheticelectron transport rate as compared to a control plant.

Embodiment 23: The modified plant of embodiment 22, wherein thephotosynthetic electron transport rate of the modified plant isincreased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% orhigher relative to the control plant.

Embodiment 24: A recombinant nucleic acid molecule comprising:

(a) one or more polynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13,15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or 47;

(b) one or more polynucleotides having at least 85%, 90%, 95% or highersequence identity to one or more of the polynucleotides set forth in(a); or

(c) a fragment of any one of the polynucleotides set forth in (a) or (b)that regulates gene expression; and

further comprising a polynucleotide heterologous to the one or morepolynucleotides of (a) or (b) or the one or more fragments of (c).

Embodiment 25: A recombinant polypeptide molecule comprising:

(a) one or more polypeptides comprising SEQ ID NOS: 87, 88, or 89;

(b) one or more polypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16,18, 20, 24, 26, 28, 30, 32, 41, 43 or 48;

(c) one or more of the polypeptides set forth in (a) having at least85%, 90%, 95% or higher sequence identity to one or more of thepolypeptides set forth in (b); or

(d) one or more fragments of any one of the polypeptides set forth in(a), (b), or (c) that regulates gene expression; and

further comprising a polypeptide heterologous to the one or morepolypeptides of (a), (b), or (c) or the one or more fragments of (d).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a CLUSTAL O(1.2.4) multiple sequence alignment of theswitchgrass STR1 transcription factor (SEQ ID NO: 2) and its maizeorthologs. SEQ IDs of proteins in alignment are as follows:GRMZM2G142179 (SEQ ID NO: 32); GRMZM2G018984 (SEQ ID NO: 10);Pavir.Ib00526 (SEQ ID NO: 36); Pavir.Ba00410 (SEQ ID NO: 2);Pavir.Bb03337 (SEQ ID NO: 33); GRMZM2G171179 (SEQ ID NO: 8);Pavir.J104875 (SEQ ID NO: 51); Pavir.Aa00281 (SEQ ID NO: 35);GRMZM2G018398 (SEQ ID NO: 4); and GRMZM2G110333 (SEQ II) NO: 6).

FIG. 2 shows a CLUSTAL O(1.2.4) multiple sequence alignment ofswitchgrass SUFI transcription factor (SEQ ID 12) and its maizeorthologs. SEQ IDs of proteins in alignment are as follows:Pavir.Aa02595 (SEQ ID NO: 12); GRMZM2G016434 (SEQ ID NO: 14);GRMZM2G457562 (SEQ ID NO: 41); GRMZM2G100727 (SEQ ID NO: 43);Pavir.J04335.1 (SEQ II) NO: 39); GRMZM2G309731 (SEQ II) NO: 20);Pavir.Gb01735.1 (SEQ ID NO: 38); GRMZM2G087059 (SEQ ID NO: 16); andGRMZM2G425798 (SEQ ID NO: 18).

FIG. 3 shows a CLUSTAL O(1.2.4) multiple sequence alignment ofswitchgrass BMY1 transcription factor (SEQ ID 22) and its maizeorthologs. SEQ IDs of proteins in alignment are as follows: Pavir.J05081(SEQ ID NO: 22); Pavir.Ba00451 (SEQ II) NO: 44); GRMZM2G384528 (SEQ IDNO: 24); GRMZM2G180947 (SEQ ID NO: 26); Pavir.Ib01924 (SEQ ID NO: 45);Pavir.Eb03638 (SEQ ID NO: 49); GRMZM2G303465 (SEQ II) NO: 48);Pavir.J02009 (SEQ II) NO: 46); Pavir.J02756 (SEQ II) NO: 50);GRMZM2G064426 (SEQ ID NO: 28); and GRMZM5G804893 (SEQ ID NO: 30).

FIG. 4 illustrates the expression pattern of select maize orthologs ofthe switchgrass transcription factors STR1, STIF1, and BMY1 in maize.A.-C. In silico analysis of the expression pattern of genes for themaize orthologs of (A) STR1 (GRMZM2G110333, SEQ ID NO: 5), (B) STIF1(GRMZM2G016434, SEQ ID NO: 13), and (C) BMY1 (GRMZM2G384528, SEQ ID NO:23) in different organs and developmental stages in maize. Data wasretrieved from the maize Electronic Fluorescent Pictograph browser(website: bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi). Levels ofexpression signals are in FPKM units (Fragment Per Kilobase of exon perMillion fragments mapped). FPKM estimates the relative transcriptabundance of each gene by combining the expression of all thetranscripts of a gene. D. Expression analysis of the maize orthologsusing RT-PCR. The levels of expression of the maize putative functionalorthologs in different organs at different developmental stages ofgreenhouse grown maize plants (inbred line B73) were analyzed.

FIG. 5 illustrates expression cassettes for overexpression of theGRMZM2G384528 gene (SEQ ID NO: 23), a maize ortholog of the switchgrassBMY1 (SEQ ID NO: 21) transcription factor. (A) An expression cassettefor YTEN26 (SEQ ID NO: 66) containing the hybrid maize cab-m5 promoterfused to the maize hsp70 intron (SEQ ID NO: 64); the maize GRMZM2G384528transcription factor gene; and the maize hsp70 terminator. (B) Anexpression cassette for YTEN27 (SEQ ID NO: 67) containing the maizeMADS-box promoter (SEQ ID NO: 56); the maize GRMZM2G384528 transcriptionfactor gene; and the maize hsp70 terminator; (C) An expression cassettefor YTEN28 (SEQ ID NO: 68) containing the maize trpA promoter (SEQ IDNO: 74); the maize GRMZM2G384528 transcription factor gene; and themaize hsp70 terminator; (D) An expression cassette for YTEN29 (SEQ IDNO: 69) containing the maize ubiquitin promoter and the maize ubiquitinintron (SEQ ID NO: 65); the maize GRMZM2G384528 transcription factorgene; and the maize hsp70 terminator.

FIG. 6 illustrates genetic components at different stages of the Casenzyme mediated genome editing process using the Cas9 enzyme as anexample. Delivery of the genetic components can be achieved in multipleways. Genetic transformation can be used to deliver the expressionconstruct depicted in (A) into a plant cell. Transcription of (A) willproduce the single guide RNA (sgRNA) depicted in (B). The sgRNA willcomplex with the Cas9 enzyme (that is delivered separately throughgenetic transformation or other means) and achieve the structuredepicted in (C) to promote cleavage of the target genomic DNA at the“guide target sequence”. Alternatively, the sgRNA (B) can be synthesizedin vitro and introduced into cells, often in the form ofRibonucleoprotein complexes (RNPs) that contain Cas9 protein to producethe structure depicted in (C) to promote cleavage of the target genomicDNA at the “guide target sequence”. When using plant transformationtechniques, the expression cassette (A) for production of the sgRNA iscomposed of a promoter, often a plant RNA polymerase III promoter, DNAencoding the “guide” of the sgRNA, DNA encoding a “guide RNA scaffold”(gRNA Sc), and a poly T-termination signal. The combination of the“guide” and the “guide RNA scaffold” are necessary to form a functionalsgRNA. The DNA encoding the guide portion of the sgRNA in (A) is oftenidentical to the “guide target sequence” of the genomic DNA to be cut in(C), however several mismatches, depending on their position, can betolerated and still promote double stranded DNA cleavage. The guideportion of the sgRNA pairs with this complementary DNA sequence to bemutated (referred to as guide target sequence #3 in figure) that isadjacent to a 3′ protospacer adjacent motif (PAM) (C), an additionalrequirement for target recognition, and double stranded DNA cleavageoccurs. When using the Cas9 enzyme for cleavage, all guide targetsequences are typically ˜20-nucleotides adjacent to a 3′ PAM sequence of(NGG) to initiate cleavage by the Cas9 enzyme. When using the CpfIenzyme for cleavage, guide target sequences are typically ˜23nucleotides adjacent to a 5′ PAM sequence that varies with the specificenzyme. PAM sequences for select CpfI enzymes including engineeredvariants are shown in TABLE 7.

FIG. 7 illustrates the strategy for promoter replacement to change theexpression pattern of a transcription factor using CRISPR genomeediting. A. Guide target sequences (˜20 nt) in genomic DNA that areadjacent to a 3′ PAM sequence of (NGG) are identified in the region ofthe endogenous promoter to be replaced. DNA cassettes encoding sgRNA(See FIG. 6) are designed to bind the genomic DNA at the identifiedguide target sequences to promote DNA cleavage and excision of thepromoter DNA. The general numbering strategy used for the promoterregion for identifying guide target sequences is as follows. Thesequence of the 5′UTR of the gene of interest plus at least anadditional 1000 bp was analyzed for guide target sequences adjacent to aPAM site to target portions of the promoter region for excision. Thisgenomic DNA sequence is given a SEQ ID number in TABLE 5 and TABLE 6.Since the length of the 5′ UTR varies for each gene, x denotes the sizeof the known or predicted 5′ UTR. Position #(1000+x) is the basedirectly in front of the ATG at the start of the coding sequence. Inthis example, guide target sequences identified for the design of threedifferent sgRNAs are depicted in the promoter region. Pairs of sgRNAscan be used to excise regions of the promoter DNA for insertion of thenew promoter replacement cassette, or alternatively, one sgRNA can beused. B. Cassettes for delivery into plant cells to achieve promoterreplacement include i. a cassette to deliver the new promoter flanked byregions homologous to each side of the nuclease cut site [left and rightflanking regions in (B), the flanking regions can additionally beflanked by guide target sequences and an adjacent PAM site to promoterelease of the cassette by Cas9 from a construct or other DNA]; ii. anexpression cassette for the Cas9 nuclease or other site specificnuclease; and iii. an expression cassette(s) for DNA encoding sgRNAs totarget cut sites that excise a portion or the whole promoter region.These cassettes can be transformed into the plant separately or on thesame DNA through a variety of plant transformation methods includingprotoplast transformation, particle bombardment, nanotube ornanoparticle mediated DNA delivery (Kwak et al., 2019, NatureNanotechnology, DOI 10.1038/s41565-019-0375-4) (Demirer et al, 2019,Nature Nanotechnology, DOI 10.1038/s41565-019-0382-5), andAgrobacterium-mediated transformation. The sgRNAs initiate aCas9-induced double stranded DNA cleavage at the guide target sequence(or sgRNA binding site) in (A), whose sequence is complementary to theguide portion of the sgRNA. The regions of the promoter insertioncassette homologous to each side of the nuclease cut site direct thecassette's insertion into genomic DNA through the plants endogenoushomology directed repair mechanism. C. Alternatively, CRISPR mediatedpromoter replacement can be achieved through the use ofRibonucleoprotein complexes (RNPs), The RNPs are created from a promoterinsertion cassette, purified Cas9 enzyme, and synthesized sgRNA1 andsgRNA3 molecules. RNPs can be created and transformed into protoplastsas previously described by Woo et al., Nature Biotechnology, 2015, 33,1162-1164. Nanoparticles or nanotubes capable of delivering biomoleculesto plants can also be used (for review see Cunningham, 2018, TrendsBiotechnol., 36, 882). D. Structure of the edited plant genomic DNAcontaining the new heterologous promoter inserted at the positions ofGuide target sequences #1 and #3, that is created through (B) genetictransformation of cassettes or (C) delivery of RNPs,

FIG. 8 illustrates the plasmid maps for insertion of a heterologousmaize promoter in front of the GRMZM2G384528 (SEQ ID NO: 23) gene, amaize ortholog of the switchgrass BMY1 transcription factor, usingCRISPR Cas mediated promoter insertion through homology directed repair.Constructs are as follows: (A) binary construct YTEN30 (SEQ ID NO: 71)for Agrobacterium-mediated transformation to deliver the maize ubiquitinpromoter and maize ubiquitin intron (SEQ ID NO: 70), (B) constructYTEN31 (SEQ ID NO: 72), a non-binary construct for transformation byprotoplast transfection or particle bombardment to deliver the maizeubiquitin promoter and maize ubiquitin intron (SEQ ID NO: 70), and (C)DNA fragment YTEN32 (SEQ ID NO: 73) for delivery of the maize ubiquitinpromoter and maize ubiquitin intron (SEQ ID NO: 70) to plant cells inribonucleoprotein complexes (RNPs). (A) The YTEN30 construct contains adouble enhanced CaMV 35S promoter driving the expression of a geneexpressing Cas9 which has been codon optimized for rice. The geneencoding Cas9 is flanked by nuclear localization sequences (NLS) toensure delivery into nuclei. The rice codon-optimized Streptococcuspyrogenes Cas9 and NLS sequence were synthesized using sequencesdescribed by Shan et al., 2013, Nat Biotechnol, 3, 686-688. ACauliflower Mosaic Virus (CaMV) terminator sequence is downstream of thegene encoding Cas9. DNA fragments encoding two guides are fused to DNAencoding the guide RNA scaffold (gRNA Sc) to encode two separatefunctional sgRNAs. The DNA fragments are labeled Guide #1 and Guide #3in the map and are equivalent to Guide target sequences #1 and #3 forGRMZM2G384528 in TABLE 5 whose positions within the promoter region areshown in FIG. 7. The resulting sgRNAs, produced upon expression of theDNA encoding the guide and gRNA Sc fragments from the rice U6 promoter(OsU6-2p), are designed to bind to the complementary guide targetsequence on the genomic DNA and excise the promoter region ofGRMZM2G384528. A poly T-termination signal is located downstream of theDNA encoding each sgRNA. A cassette containing the promoter to beinserted in the double stranded break created by the Cas9 nuclease nearthe PAM sites adjacent to guide target sequences #1 and #3 contains thefollowing elements: DNA corresponding to the Guide #3 target sequenceand its associated PAM sequence (labeled BMY1-3); a DNA fragment (˜800bp in length) that is homologous to the region flanking the left side ofthe genomic DNA cut site (labeled HR-L); the maize ubiquitin promotersequence with an intron (SEQ ID 70); a DNA fragment (˜800 bp in length)that is homologous to the region flanking the right side of the genomicDNA cut site (labeled HR-R); and DNA corresponding to the Guide #1target sequence and its associated PAM sequence (labeled BMY1-1). Thehomologous regions flanking the promoter to be inserted enable insertionof the fragment into the plant's genomic DNA by the plant's homologydirected repair mechanism. An expression cassette for selection oftransgenic plants is included in the vector and contains a doubleenhanced CaMV35S promoter, an hsp70 intron, a hptI gene encodinghygromycin phosphotransferase containing an intron from the beancatalase-1 gene (CAT-1 intron), and a CaMV35S polyA sequence to providehygromycin resistance to transgenic plants. The T-DNA sequence forinsertion into the plant by Agrobacterium-mediated transformation isflanked by T-DNA left and right border sequences. (B) The YTEN31construct is similar to YTEN30, except it is not a binary vector anddoes not have T-DNA border sequences. (C) The YTEN 32 fragment containsonly the promoter insertion cassette of vectors YTEN30 and YTEN31. It isintended for use in RNPs with purified Cas9 enzyme and synthesizedsgRNAs to cleave at the Guides #1 and #3 target sequences in the genomicDNA.

FIG. 9 illustrates cassettes for insertion into the genome at a Casnuclease cleavage site to modulate the level of expression of thetranscription factor. A. Schematic of the plant genomic DNA to bemodified showing the positioning of three guide target DNA sequences (a,b, and c). The guide target sequences are adjacent to PAM sequences. B.Cassettes to be inserted to modulate expression of the transcriptionfactor can be selected from one or more of the following: i. anexpression cassette for a second copy of a transcription factor ofinterest containing a heterologous promoter (designated promoter x), thecoding sequence (CDS) of the transcription factor, and a 3′ UTR(designated 3′UTR X). In this example, the insertion of this cassette istargeted to a genomic region where an sgRNA capable of binding to guidetarget sequence a will initiate a Cas9-induced double stranded DNAcleavage. The promoter insertion cassette is flanked by regionshomologous to each side of the nuclease cut site to direct thecassette's insertion through the plant's endogenous homology directedrepair mechanism. ii. a cassette for insertion of an intron between thepromoter and the start codon of the gene. In this example, the insertionof the intron cassette is targeted to a genomic region where an sgRNAcapable of binding to guide target sequence b will initiate aCas9-induced double stranded DNA cleavage in a region near the 5′ UTRand the start codon of the transcription factor gene. The introninsertion cassette is flanked by regions homologous to each side of thenuclease cut site to direct the cassettes insertion through the plantsendogenous homology directed repair mechanism. iii. a cassette forinsertion of a promoter enhancer upstream of the endogenous promoter. Inthis example, the insertion of the enhancer cassette is targeted to agenomic region where an sgRNA capable of binding to guide targetsequence c will initiate a Cas9-induced double stranded DNA cleavage.The enhancer insertion cassette is flanked by regions homologous to eachside of the nuclease cut site to direct the cassette's insertion throughthe plant's endogenous homology directed repair mechanism. C.Illustration of the products of site-directed insertion for cassette i,ii, and/or iii into genomic DNA. While the illustration shows insertionof all three cassettes, one skilled in the art will understand thatinsertion(s) can be selected from one or more cassettes.

DETAILED DESCRIPTION OF THE INVENTION

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein we use the terms “crops” and “plants” interchangeably.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” or “recombinant expressionconstruct”, which are used interchangeably, refers to any gene that isnot a native gene, comprising regulatory and coding sequences that arenot found together in nature. A “Cis-genic gene” is a chimeric genewhere the DNA sequences making up the gene are from the same plantspecies or a sexually compatible plant species where the cis-genic geneis deployed in the same species from which the DNA sequences wereobtained. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature.“Endogenous gene” refers to a native gene in its natural location in thegenome of an organism. A “foreign” gene refers to a gene not normallyfound in the host organism, but that is introduced into the hostorganism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure. As used herein the term “coding sequence” refers to a DNAsequence which codes for a specific amino acid sequence. “Regulatorysequences” refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include, but are not limited to, promoters, translationleader sequences, introns, and polyadenylation recognition sequences. Asused herein “gene” includes protein coding regions of the specific genesand the regulatory sequences both 5′ and 3′ which control the expressionof the gene.

As used herein a “modified plant” refers to non-naturally occurringplants or crops engineered as described throughout herein.

As used herein a “control plant” means a plant that has not beenmodified as described in the present disclosure to impart an enhancedtrait or altered phenotype. A control plant is used to identify andselect a modified plant that has an enhanced trait or altered phenotype.For instance, a control plant can be a plant that has not been modifiedor has not been genome edited to express or to inhibit its endogenousgene product. A suitable control plant can be a non-transgenic plant ofthe parental line used to generate a transgenic plant, for example, awild type plant devoid of a recombinant DNA. A suitable control plantcan also be a transgenic plant that contains recombinant DNA thatimparts other traits, for example, a transgenic plant having enhancedherbicide tolerance. A suitable control plant can in some cases be aprogeny of a hemizygous transgenic plant line that does not contain therecombinant DNA, known as a negative segregant, or a negative isogenicline.

As used herein the terms “biomass yield” or “biomass content” refer toincrease or decrease in the % dry weight in an amount greater than anotherwise identical plant, cultured under identical conditions, butlacking any corresponding modification, e.g., gene editing or thetransgene in a control plant.

As used herein, the terms “increase activity”, “increase expression” or“upregulated” are used interchangeably and mean the activity of thetranscription factor is increased or higher than the expression of thesame gene in the same plant species before the gene was modified asdescribed herein. The term also encompasses the situation where theactivity of the transcription factor gene is upregulated in a tissue orat a stage of plant development as compared to the activity of thetranscription factor gene in the tissue or developmental stage beforethe gene was modified. Upregulation should be understood to include anincrease in the level or activity of a target gene in a cell and/or anincrease in the expression of a particular target polypeptide in a cellwhich normally expresses the target polypeptide. For instance, a 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold,10-fold, 20-fold, 50-fold, 100-fold increase in the level of activity ofa target polypeptide in the cell. With respect to term “2-foldincrease”, “upregulated 2-fold” and 100% increase is usedinterchangeably.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for increased expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity). When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percent sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percent sequence identity” means the value determinedby comparing two aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percent sequence identity.

The term “corn plant” includes whole plant, mature plants, seeds, shootsand seedlings, and parts, propagation material, plant organ tissue,protoplasts, callus and other cultures, for example cell cultures,derived from corn plants. The term “mature corn plants” refers to plantsat any developmental stage beyond the seedling. The term “seedlings”refers to young, immature plants at an early developmental stage.

PREFERRED EMBODIMENTS

The present disclosure relates to transcription factor genes in cornwhose expression or activity can be modulated to increase corn cropperformance and corn crops having increased expression of thetranscription factor genes which have improved performance compared tothe same corn plants with normal expression levels of these genes. Alsodisclosed are specific corn transcription factor gene sequences, DNAsequences, RNA sequences and materials and methods for modifying plantcells and plants such that they have increased expression of thetranscription factor genes, methods for identifying corn plant cells andcorn plants with increased expression of the transcription factor genesand methods for producing fertile corn plants with increased expressionof the transcription factor genes wherein the modified corn plants haveimproved performance as compared to the same corn plants before theywere modified to increase the expression of these genes.

In various aspects, the present invention provides corn transcriptionfactors and genes encoding the corn transcription factors useful forpracticing the disclosed invention and include those that can functionas positive controllers in corn plants. Transcription factors functionto either increase the activity of specific metabolic pathways or generegulatory networks in plants or to decrease them. Herein we disclosecorn transcription factors and genes encoding the corn transcriptionfactors that function as positive controllers in corn and whoseincreased expression in corn is important for improved performance.

In one embodiment, the corn transcription factors comprise (a) one ormore polypeptides comprising SEQ ID NOS: 87, 88, or 89. These sequencescorrespond to consensus sequences for three groups of corn transcriptionfactors that function as positive controllers in corn and whoseincreased expression in corn is important for improved performance. Insome examples, the corn transcription factors comprise (b) one or morepolypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16, 18, 20, 24, 26,28, 30, 32, 41, 43 or 48. Also in some examples, the corn transcriptionfactors comprise (c) one or more of the polypeptides set forth in (a)having at least 85%, 90%, 95% or higher sequence identity to one or moreof the polypeptides set forth in (b). Also in some examples, the corntranscription factor genes comprise (d) one or more polynucleotidescomprising SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, 29, 31,40, 42 or 47. Also in some examples, the corn transcription factor genescomprise (e) one or more polynucleotides having at least 85%, 90%, 95%or higher sequence identity to one or more of the polynucleotides setforth in (d). Also, in some examples, the corn transcription factorscomprise (f) one or more polypeptides encoded by one or more of thepolynucleotides set forth in (d) or (e).

Thus, in one example the corn transcription factor comprises one or moreof SEQ ID NOS: 4, 8, 10, 16, 18, 20, 26, 28, 30, 32, 41, 43, or 48.

In another example the corn transcription factor comprises one or moreof SEQ ID NO: 6 (GRMZM2G110333), SEQ ID NO: 14 (GRMZM2G016434), and SEQIO NO: 24 (GRMZM2G384528).

The present invention provides isolated nucleic acid molecules for genesencoding transcription factors, and variants thereof. Exemplaryfull-length nucleic acid sequences for genes encoding transcriptionfactors and the corresponding amino acid sequences are presented inTABLE 1 and TABLE 2. The nucleic acid sequence can be preferably greaterthan 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to thewild-type gene.

In another embodiment, the nucleic acid molecule of the presentinvention encodes a polypeptide having an amino acid sequence disclosedin TABLE 1 or TABLE 2. Preferably, the nucleic acid molecule of thepresent invention encodes a polypeptide sequence having at least 85%,90% or 95% identity to the amino acid sequences shown in TABLE 1 orTABLE 2 and the identity can even more preferably be 96%, 97%, 98%, 99%,99.9% or even higher.

According to another aspect of the present invention, isolatedpolypeptides (including muteins, allelic variants, fragments,derivatives, and analogs) encoded by the nucleic acid molecules of thepresent invention are provided. In one embodiment, the isolatedpolypeptide comprises the polypeptide sequence corresponding to apolypeptide sequence shown in TABLE 1 or TABLE 2.

In an alternative embodiment of the present invention, the isolatedpolypeptide comprises a polypeptide sequence at least 85%, 90%, 95% orhigher sequence identity to a polypeptide sequence shown in TABLE 1 orTABLE 2. Preferably the isolated polypeptide of the present inventionhas at least 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%,98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9% or even higher identity to a polypeptide ofSEQ ID NOS: 4, 6, 8, 10, 14, 16, 18, 20, 24, 26, 28, 30, 32, 41, 43 or48.

The different families of transcription factors found in crops aredescribed for example by Lin, et. al., (2014, BMC Genomics, 15,818-820).

The modern corn genome contains around 39,000 thousand genes and about2,500 of these are transcription factors (Lin, et. al., 2014, BMCGenomics, 15, 818-820). It is known that many plant species contain morethan one copy of a specific gene and this invention encompasses allcopies of the specific genes identified.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Computerimplementations of these mathematical algorithms can be utilized forcomparison of sequences to determine sequence identity. Suchimplementations include, but are not limited to: CLUSTAL in the PC/Geneprogram (available from Intelligenetics, Mountain View, Calif.); theALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTAin the GCG Wisconsin Genetics Software Package, Version 10 (availablefrom Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA).Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. BLASTP protein searches can beperformed using default parameters. See,blast.ncbi.nlm.nih.gov/Blast.cgi.

Sequence alignments and percent similarity calculations may bedetermined using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.) or using the AlignXprogram of the Vector NTI bioinformatics computing suite (Invitrogen,Carlsbad, Calif.). Multiple alignment of the sequences are performedusing the Clustal method of alignment (Higgins and Sharp, CABIOS5:151-153 (1989)) with the default parameters (GAP PENALTY=10, GAPLENGTH PENALTY=10). Default parameters for pairwise alignments andcalculation of percent identity of protein sequences using the Clustalmethod are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Fornucleic acids these parameters are GAP PENALTY=10, GAP LENGTHPENALTY=10, KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. A“substantial portion” of an amino acid or nucleotide sequence comprisesenough of the amino acid sequence of a polypeptide or the nucleotidesequence of a gene to afford putative identification of that polypeptideor gene, either by manual evaluation of the sequence by one skilled inthe art, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Altschul, S. F. et al., J. Mol. Biol.215:403-410 (1993)) and Gapped Blast (Altschul, S. F. et al., NucleicAcids Res. 25:3389-3402 (1997)). BLASTN refers to a BLAST program thatcompares a nucleotide query sequence against a nucleotide sequencedatabase.

Disclosed herein are corn (maize) transcription factor genes specifiedby SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, 29, 31, 40, 42 or47, and methods for increasing their expression alone or in combinationsin corn to improve corn performance are included in the scope of thisinvention.

Based on the disclosure herein, it will be apparent to a person of skillin the art how to use the genes and the proteins encoded by the genesidentified by SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, 29,31, 40, 42 or 47 by different methods to increase the expression of oneor more of the transcription factor genes in corn such that theperformance of the corn crop is improved.

In some embodiments, the polynucleotide is upregulated by usingtraditional genetic engineering methods which are well known in the artand have recently been reviewed by Qiudeng Que*, Sivamani Elumalai,Xianggan Li, Heng Zhong, Samson Nalapalli, Michael Schweiner, XiaoyinFei, Michael Nuccio, Timothy Kelliher, Weining Gu, Zhongying Chen, andMary-Dell M. Chilton (2014) Frontiers in Plant Science 5, article 379,pp 1-19.

In some embodiments, the polynucleotide is upregulated by the use of newbreeding techniques where targeted DNA sequence changes are facilitatedthru the use of Zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2 andZFN-3, see U.S. Pat. No. 9,145,565, incorporated by reference in itsentirety), Oligonucleotide directed mutagenesis (ODM), Cisgenesis andintragenesis, RNA-dependent DNA methylation (RdDM, which does notnecessarily change nucleotide sequence but can change the biologicalactivity of the sequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration (agro-infiltration “sensu stricto”, agro-inoculation,floral dip), Transcription Activator-Like Effector Nucleases (TALENs,see U.S. Pat. Nos. 8,586,363 and 9,181,535, incorporated by reference intheir entireties), the CRISPR/Cas system (see U.S. Pat. Nos. 8,697,359;8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308;8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641), engineeredmeganuclease re-engineered homing endonucleases, DNA guided genomeediting (Gao et al., Nature Biotechnology (2016), doi: 10.1038/nbt.3547,incorporated by reference in its entirety), and synthetic genomics. Acomplete description of each of these techniques can be found in thereport made by the Joint Research Center (JRC) Institute for ProspectiveTechnological Studies of the European Commission in 2011 and titled “Newplant breeding techniques—State-of-the-art and prospects for commercialdevelopment” website:ipts.jrc.ec.europa.eu/publications/pub.cfm?id=4100).

Modulation of candidate transcription factor genes are performed throughknown techniques in the art, such as without limitation, by geneticmeans, enzymatic techniques, chemicals methods, or combinations thereof.Activation may be conducted at the level of DNA, mRNA or protein, andinhibit the expression of one or more candidate transcription factorgenes or the corresponding activity. Preferred activation methods affectthe expression of the transcription factor gene and lead to the increaseof gene product in the plant cells. Increased expression can be obtainedvia mutagenesis of the transcription factor gene. For example, amutation in the coding sequence can induce, depending upon the nature ofthe mutation, increased activity of the protein; a mutation at orintroduction of a splicing site can also increase expression andactivity; a mutation in the promoter sequence can increase its activityand increase expression of the transcription factor gene. Mutagenesiscan be performed, e.g., to modify the promoter, or by inserting anexogenous sequence, e.g., a transcription enhancer or intron, into saidpromoter. It can also be performed by inducing point mutations, e.g.,using ethyl methanesulfonate (EMS) mutagenesis or radiation. The mutatedalleles can be detected, e.g., by PCR, by using specific primers of thegene. Rodriguez-Leal et al. describe a promoter editing method thatgenerates a pool of promoter variants that can be screened to evaluatetheir phenotypic impact (Rodriguez-Leal et al., 2017, Cell, 171, 1-11).This method can be incorporated into the present invention to upregulatenative promoters of transcription factors of interest.

Various high-throughput mutagenesis and splicing methods are describedin the prior art. By way of examples, we may cite “TILLING” (TargetingInduced Local Lesions In Genome)-type methods, described by Till, Comaiand Henikoff (2007) (R. K. Varshney and R. Tuberosa (eds.),Genomics-Assisted Crop Improvement: Vol. 1: Genomics Approaches andPlatforms, 333-349).

Corn plants comprising a mutation in the candidate transcription factorgenes that increase the activity or stability of the protein product arealso part of the goal of the present invention. This mutation can be,e.g., may be a point mutation of said coding sequence or of saidpromoter.

Enhanced expression of the transcription factor proteins can also beobtained by gene editing of the candidate genes. Examples of methods forediting genes in corn have recently been published (Svitashev, S.,Young, J. K., Schwartz, C., Gao, H., Falco, S. C. and Cigan, A. M. 2015,Methods for targeted mutagenesis, precise gene editing, andsite-specific gene insertion in maize using Cas9 and guide RNA. PlantPhysiology 169, 931-945). Various methods can be used for gene editing,by using transcription activator-like effector nucleases (TALENs),clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9)or zinc-finger nucleases (ZFN) techniques (as described in Belhaj et al,2013, Plant Methods, vol 9, p 39, Chen et al, 2014 Methods Volume 69,Issue 1, p 2-8). Preferably, the enhancement of a transcription factorprotein, or the enhancement of its expression, is obtained by usingClustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9)or CRISPR/Cpf1. The use of this technology in genome editing is welldescribed in the art, for example in Fauser et al. (Fauser et al, 2014,The Plant Journal, Vol 79, p 348-359), and references cited herein. Inshort, CRISPR is a microbial nuclease system involved in defense againstinvading phages and plasmids. CRISPR loci in microbial hosts contain acombination of CRISPR-associated (Cas) genes as well as non-coding RNAelements capable of programming the specificity of the CRISPR-mediatednucleic acid cleavage (sgRNA). At least classes (Class I and II) and sixtypes (Types I-VI) of Cas proteins have been identified across a widerange of bacterial hosts. One key feature of each CRISPR locus is thepresence of an array of repetitive sequences (direct repeats)interspaced by short stretches of non-repetitive sequences (spacers).The non-coding CRISPR array is transcribed and cleaved within directrepeats into short crRNAs containing individual spacer sequences(protospacers), which direct Cas nucleases to the target site. The TypeII CRISPR/Cas is one of the most well characterized systems and carriesout targeted DNA double-strand break in four sequential steps. First,two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribedfrom the CRISPR locus. Second, tracrRNA hybridizes to the repeat regionsof the pre-crRNA and mediates the processing of pre-crRNA into maturecrRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crickbase-pairing between the spacer on the crRNA and the protospacer on thetarget DNA next to the protospacer adjacent motif (PAM), an additionalrequirement for target recognition. Finally, Cas9 mediates cleavage oftarget DNA to create a double-stranded break within the protospacer.Cas9 is thus the hallmark protein of the Type II CRISPR-Cas system, anda large monomeric DNA nuclease guided to a DNA target sequence adjacentto the PAM (protospacer adjacent motif) sequence motif by a complex oftwo noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA(tracrRNA). The Cas9 protein contains two nuclease domains homologous toRuvC and HNH nucleases. The HNH nuclease domain cleaves thecomplementary DNA strand whereas the RuvC-like domain cleaves thenon-complementary strand and, as a result, a blunt cut is introduced inthe target DNA.

Engineered systems utilize heterologous expression of Cas9 together witha single guide RNA (sgRNA), a synthetic fusion between a crRNA and partof the tracrRNA sequence, to introduce site-specific double strandbreaks (DSBs) into genomic DNA of live cells from various organisms. Forapplications in eukaryotic organisms, codon optimized versions of Cas9,which is originally from the bacterium Streptococcus pyogenes, have beenused. The sgRNA forms a complex with the Cas9 nuclease. The “guide”portion of the sgRNA (FIG. 6), which is about 20 nucleotides in lengthand located at the 5′ end of the sgRNA, is designed to be complementaryto a DNA target sequence adjacent to a PAM sequence and confers DNAtarget specificity. Therefore, by modifying the sequence of the guideportion of the sgRNA, it is possible to create sgRNAs with differenttarget specificities. The canonical length of the guide of the sgRNA is˜20 bp. In plants, sgRNAs have been expressed using plant RNA polymeraseIII promoters, such as U6 and U3. Cas9 expression plasmids for use inthe methods of the invention can be constructed as described in the art.

The increased expression in modified engineered plants or plant cellscan be verified based on the phenotypic characteristics of theiroffspring; homozygous plants or plant cells for a mutation increasingthe expression of the transcription factor gene have a content of geneproduct that is higher than that of the wild plants (not carrying themutation in the gene) from which they originated. Alternatively, adesirable phenotypic characteristic such as photosynthesis rate,photosynthetic electron transport rate, biomass yield, seed yield, orseed oil content is measured and is at least 10% higher, preferably atleast 20% higher, at least preferably 30% higher, preferably at least40% higher, preferably at least 50% higher than that of the controlplants from which they originated. Photosynthetic parameters, such asphotochemical quantum yield (Y), non-photochemical quenching (NPQ), andelectron transport rate (ETR) can be measured in plants usingcommercially available machines, such as the Dual-PAM-100 MeasuringSystem (Heinz Walz Gmbh, Effeltrich, Germany). Increases in Y in plantsrepresent increases in the portion of absorbed quanta that is convertedinto chemically fixed energy by the photosystem I (PSI) and photosystemII (PSII) reaction centers. The photosynthetic electron transport ratesare often referred to as the electron transport rates of PSI and PSII.Increases in the electron transport rate of PSII (indicative of the rateof non-cyclic electron transfer), and the electron transport rate of PSI(indicative of both cyclic and non-cyclic electron transfer), can bedetermined. NPQ is a mechanism that plants use to protect themselvesfrom high light intensity and manipulation of NPQ can increase yield(Hubbart et al., 2018, Nature Communications Biology, 1, Article 22).

More preferably, seed yield is at least 5%, at least 10%, at least 20%,at least 40%, at least 60% higher, at least 70% higher, at least 80%higher, at least 90% higher than that of the control plants from whichthey originated. More preferably, seed yield or seed oil content is atleast 100% higher, at least 150% higher, at least 200% higher than thatof the control plants from which they originated.

The expression of the target gene or genes in the crops of interest canbe increased by any method known in the art, including the transgenebased expression of the gene or through genome editing or mutagenesis tomodify the DNA sequence of the promoter sequences of the genes disclosedherein directly in the plant cell chromosome.

Genome editing is a preferred method for practicing this invention. Asused herein the terms “genome editing,” “genome edited”, and “genomemodified” are used interchangeably to describe plants with specific DNAsequence changes in their genomes wherein those DNA sequence changesinclude changes of specific nucleotides, the deletion of specificnucleotide sequences or the insertion of specific nucleotide sequences.

As used herein “method for genome editing” includes all methods forgenome editing technologies to precisely remove genes, gene fragments,or to insert new DNA sequences into genes, to alter the DNA sequence ofcontrol sequences or protein coding regions to reduce or increase theexpression of target genes in plant genomes (Belhaj, K. 2013, PlantMethods, 9, 39; Khandagale & Nadal, 2016, Plant Biotechnol Rep, 10,327). Preferred methods involve the in vivo site-specific cleavage toachieve double stranded breaks in the genomic DNA of the plant genome ata specific DNA sequence using nuclease enzymes and the host plant DNArepair system. There are multiple methods to achieve double strandedbreaks in genomic DNA, and thus achieve genome editing, including theuse of zinc finger nucleases (ZFN), transcription activator-likeeffector nucleases (TALENs), engineered meganucleases, and theCRISPR/Cas system (CRISPR is an acronym for clustered, regularlyinterspaced, short, palindromic repeats and Cas an abbreviation forCRISPR-associated protein) (for review see Khandagal & Nadal, PlantBiotechnol Rep, 2016, 10, 327). US Patent Application 2016/0032297 toDupont describes these methods in detail. In some cases, the sequencespecificity for the target gene in the plant genome is dependent onengineering specific nucleases like zinc finger nucleases (ZFN), whichinclude an engineered DNA-binding zinc finger domain linked to anon-specific endonuclease domain such as FokI, or Tal effector nuclease(TALENS) to recognize the target DNA sequence in the plant genome. TheCRISPR/Cas genome editing system is a preferred method because of itssequence targeting flexibility. This technology requires a source of theCas enzyme and a sgRNA containing a short guide (˜20 bp), with sequencecomplementarity to the target DNA sequence in the plant genome.Depending on the type of Cas enzyme, alternatively a DNA, an RNA/DNAhybrid, or a double stranded DNA guide polynucleotide can be used. Theguide portion of this guide polynucleotide directs the Cas enzyme to thedesired cut site for cleavage with a recognition sequence for bindingthe Cas enzyme. As used herein the term Cas nuclease includes anynuclease which site-specifically recognizes CRISPR sequences based onguide RNA or DNA sequences and includes Cas9, Cpf1 and others describedbelow. CRISPR/Cas genome editing, is a preferred way to edit the genomesof complex organisms (Sander & Joung, 2013, Nat Biotech, 2014, 32, 347;Wright et al., 2016, Cell, 164, 29) including plants (Zhang et al.,2016, Journal of Genetics and Genomics, 43, 151; Puchta, H., 2016, PlantJ., 87, 5; Khandagale & Nadaf, 2016, PLANT BIOTECHNOL REP, 10, 327). USPatent Application 2016/020822 to Dupont has an extensive description ofthe materials and methods useful for genome editing in plants using theCRISPR/Cas9 system and describes many of the uses of the CRISPR/Cas9system for genome editing of a range of gene targets in crops.

There are many variations of the CRISPR/Cas system that can be used forthis technology including the use of wild-type Cas9 from Streptococcuspyogenes (Type II Cas) (Barakate & Stephens, 2016, Frontiers in PlantScience, 7, 765; Bortesi & Fischer, 2015, Biotechnology Advances 5, 33,41; Cong et al., 2013, Science, 339, 819; Rani et al., 2016,Biotechnology Letters, 1-16; Tsai et al., 2015, Nature biotechnology,33, 187), the use of a Tru-gRNA/Cas9 in which off-target mutations weresignificantly decreased (Fu et al., 2014, Nature biotechnology, 32, 279;Osakabe et al., 2016, Scientific Reports, 6, 26685; Smith et al., 2016,Genome biology, 17, 1; Zhang et al., 2016, Scientific Reports, 6,28566), a high specificity Cas9 (mutated S. pyogenes Cas9) with littleto no off target activity (Kleinstiver et al., 2016, Nature 529, 490;Slaymaker et al., 2016, Science, 351, 84), the Type I and Type III CasSystems in which multiple Cas proteins need to be expressed to achieveediting (Li et al., 2016, Nucleic acids research, 44:e34; Luo et al.,2015, Nucleic acids research, 43, 674), the Type V Cas system using theCpf1 enzyme (Kim et al., 2016, Nature biotechnology, 34, 863; Toth etal., 2016, Biology Direct, 11, 46; Zetsche et al., 2015, Cell, 163,759), DNA-guided editing using the NgAgo Argonaute enzyme fromNatronobacterium gregoryi that employs guide DNA (Xu et al., 2016,Genome Biology, 17, 186), and the use of a two vector system in whichCas9 and sgRNA expression cassettes are carried on separate vectors(Cong et al., 2013, Science, 339, 819). A unique nuclease Cpf1, analternative to Cas9 has advantages over the Cas9 system in reducingoff-target edits which creates unwanted mutations in the host genome.Examples of crop genome editing using the CRISPR/Cpf1 system includerice (Tang et. al., 2017, Nature Plants 3, 1-5; Wu et. al., 2017,Molecular Plant, Mar. 16, 2017) and soybean (Kim et., al., 2017, NatCommun. 8, 14406).

Methods for constructing the genome modified corn plant cells and cornplants include introducing into plant cells a vector comprising a geneexpression construct of one or more of the corn transcription factorgenes and a second gene expression construct comprising a selectablemarker gene.

Methods for constructing the genome modified plant cells and plantsinclude introducing into plant cells a site-specific nuclease to cleavethe plant genome at the target site or target sites and the guidesequences. Modification to the DNA sequence at the cleavage site thenoccurs through the plant cell's natural DNA repair processes. In apreferred case using the CRISPR system the target site in the plantgenome is determined by providing single guide RNA (sgRNA) sequences.

A “guide polynucleotide” also relates to a polynucleotide sequence thatcan form a complex with a Cas endonuclease and enables the Casendonuclease to recognize and optionally cleave a DNA target site. Theguide polynucleotide can be a single molecule (i.e. a single guide RNA(sgRNA) that is a synthetic fusion between a crRNA and part of thetracrRNA sequence) or a two molecules (i.e. the crRNA and tracrRNA asfound in natural Cas9 systems in bacteria). The guide polynucleotidesequence can be provided as an RNA sequence or can be transcribed from aDNA sequence to produce an RNA sequence. The guide polynucleotidesequence can also be provided as a combination RNA-DNA sequence (see forexample, Yin, H. et al., 2018, Nature Chemical Biology, 14, 311).

As used herein “guide RNA” sequences comprise a variable targetingdomain, called the “guide”, complementary to the target site in thegenome, and an RNA sequence that interacts with the Cas9 or Cpf1endonuclease, called the “guide RNA scaffold”. A guide polynucleotidethat solely comprises ribonucleic acids is also referred to as a “guideRNA”.

As used herein the “guide target sequence” refers to the sequence of thegenomic DNA adjacent to a PAM site, where the sgRNA will bind to cleavethe DNA. The “guide target sequence” is often complementary to the“guide” portion of the sgRNA, however several mismatches, depending ontheir position, can be tolerated and still allow Cas mediated cleavageof the DNA.

The method also provides introducing single guide RNAs (sgRNAs) intoplants. The single guide RNAs (sgRNAs) include nucleotide sequences thatare complementary to the target chromosomal DNA. The sgRNAs can be, forexample, engineered single chain guide RNAs that comprise a crRNAsequence (complementary to the target DNA sequence) and a commontracrRNA sequence, or as crRNA-tracrRNA hybrids. The sgRNAs can beintroduced into the cell or the organism as a DNA with an appropriatepromoter, as an in vitro transcribed RNA, or as a synthesized RNA. Basicguidelines for designing the guide RNAs for any target gene of interestare well known in the art as described for example by Brazelton et al.(Brazelton, V. A. et al., 2015, GM Crops & Food, 6, 266-276) and Zhu(Zhu, L. J. 2015, Frontiers in Biology, 10, 289-296).

Target Sequence for Increasing Expression

Examples of mutations that may lead to increased activity of thetranscription factor protein are mutations to the coding sequence thatgive rise to amino acid changes in the encoded protein.

In certain preferred embodiments, the guide polynucleotide/Casendonuclease system can be used to allow for the insertion of a promoteror promoter element of any one the transcription factor sequences of theinvention, wherein the promoter insertion (or promoter element deletion)results in any one of the following or any one combination of thefollowing: a permanently activated gene locus, an increased promoteractivity (increased promoter strength), an increased promoter tissuespecificity, a decreased promoter tissue specificity, a new promoteractivity, an extended window of gene expression, a modification of thetiming or developmental progress of gene expression, a mutation of DNAbinding elements and/or an addition of DNA binding elements. Promoterelements to be deleted can be, but are not limited to, promoter coreelements, promoter enhancer elements or 35 S enhancer elements (CaMV35Senhancers (Benfey et al, EMBO J, August 1989; 8(8): 2195-2202)). Thepromoter or promoter fragment to be deleted can be endogenous,artificial, pre-existing, or transgenic to the cell that is beingedited. Preferably the promoter element is endogenous to the cell thatis being edited

In yet another embodiment, the genomic sequence of interest to bemodified is an intron site of any one of the transcription factorsequences of the invention, wherein the modification consists ofinserting an intron enhancing motif into the intron which results inmodulation of the transcriptional activity of the gene comprising saidintron.

In a further embodiment, methods provide for modifying alternativesplicing sites of any one of the transcription factor sequences of theinvention resulting in enhanced production of the functional genetranscripts and gene products (proteins).

In additional embodiments, the modification of the transcription factorsequences of the invention include editing the intron borders ofalternatively spliced genes to alter the accumulation of splicevariants.

In other embodiments, the guide polynucleotide/Cas endonuclease systemcan be used to modify or replace a coding sequence of the transcriptionfactor in the genome of a plant cell, wherein the modification orreplacement results in any one of the following, or any one combinationof the following: an increased protein activity, an increased proteinfunctionality, a site specific mutation, a protein domain swap, aprotein knock-out, a new protein functionality, a modified proteinfunctionality.

The guide RNA/Cas endonuclease system can be used to allow for theinsertion of a promoter element to increase the expression of thetranscription factor sequences of the invention. Promoter elements, suchas enhancer elements, are often introduced in promoters driving geneexpression cassettes in multiple copies for trait gene testing or toproduce transgenic plants expressing specific traits. Enhancer elementscan be, but are not limited to, a 35S enhancer element (Benfey et al,EMBO J, August 1989; 8(8): 2195-2202). In some plants (events), theenhancer elements can cause a desirable phenotype, a yield increase, ora change in expression pattern of the trait of interest that is desired.It may be desired to remove the extra copies of the enhancer elementwhile keeping the trait gene cassettes intact at their integratedgenomic location. The guide RNA/Cas endonuclease can be used to removethe unwanted enhancing element from the plant genome. A guide RNA can bedesigned to contain a variable targeting region targeting a target sitesequence of 12-30 bps adjacent to a NGG (PAM) in the enhancer. The Casendonuclease can make cleavage to insert one or multiple enhancers. Theguide RNA/Cas endonuclease system can be introduced by eitherAgrobacterium or particle gun bombardment. Alternatively, nanotube ornanoparticle mediated DNA delivery (Kwak et al., 2019, NatureNanotechnology, DOT 10.1038/s41565-019-0375-4) (Demirer et al, 2019,Nature Nanotechnology, DOI 10.1038/s41565-019-0382-5) can be used. Twodifferent guide RNAs (targeting two different genomic target sites) canbe used to remove multiple enhancer elements from the genome of a plant.

In some embodiments, the genome modified plant has improved performanceas compared to a plant of the same type which does not have the genomemodification. The improved performance of the genome modified plantincludes for example, higher photosynthesis rates, higher photosyntheticelectron transport rate, higher non-photochemical quenching, reducedphotorespiration rates, higher biomass yield or content, higher seedyield, improved harvest index, higher seed oil content, improvednutritional composition, improved nitrogen use efficiency, droughtresistance, flood resistance, disease resistance, salt tolerance, higherCO₂ assimilation rate, or lower transpiration rate. The genome modifiedplant can have a CO₂ assimilation rate that is higher than for acorresponding reference plant not comprising the genome modification.For example, the genome modified plant can have a CO₂ assimilation ratethat is at least 5% higher, at least 10% higher, at least 20% higher, atleast 40% higher, at least 60% higher, at least 100% higher, at least200% higher or at least 400% higher than for a corresponding referenceplant not comprising the genome modification.

The genome modified plant can also have a transpiration rate that islower than for a corresponding reference plant not comprising the genomemodification. For example, the genome modified plant can have atranspiration rate that is at least 5% lower, at least 10% lower, atleast 20% lower, at least 40% lower, at least 60% lower or at least 100%lower than for a corresponding reference plant not comprising the genomemodification.

The genome modified plant can have a seed yield or a seed oil contentthat is higher than for a corresponding reference plant not comprisingthe genome modification. For example, the genome modified plant can havea seed yield or seed oil content that is at least 5% higher, at least10% higher, at least 20% higher, at least 40% higher, at least 60%higher, at least 80% higher or at least 100% higher, than for acorresponding reference plant not comprising the genome modification.

The genome modified plant can have a seed yield that is higher than fora corresponding reference plant not comprising the genome modification.For example, the genome modified plant can have a seed yield that is atleast 5% higher, at least 10% higher, at least 20% higher, at least 40%higher, at least 60% higher, at least 80% higher or at least 100%higher, than for a corresponding reference plant not comprising thegenome modification.

Plants of Interest

Transcription factor genes, including specific corn transcription factorgene sequences are useful as targets for upregulation, alone or incombinations, to improve corn crop performance are described herein.Preferably the transcription factor genes are upregulated in an inbredcorn line to reduce the time for development and testing of the impactof the upregulated transcription factor in corn hybrids. Methods ofupregulating the transcription factor genes in corn include transgenicapproaches and the use of site-specific nucleases, guide RNAs, guideRNA-DNA hybrids and guide DNAs. DNA constructs useful in the methods aredescribed herein. Methods for introducing either the genetic constructor the site-specific nuclease and guide RNAs into plant cells and planttissues are also described herein and methods for identifying plantcells, plant tissue and fertile plants having increased expression ofthe transcription factor genes made using these methods are disclosedherein. As used herein, “transgenic” refers to an organism in which anucleic acid fragment containing a heterologous or “non-native”nucleotide sequence has been introduced. Preferably the non-nativenucleotide sequence is derived from nucleotide sequences naturallypresent in corn. The increased expression of the transcription factorsintroduced into the plants are stable, inheritable and impart improvedplant performance.

Modified Plant Genomes Using CRISPR/Cas, Guide RNAs

Examples of simultaneous CRISPR/Cas9 or CRISPR/Cpf1 gene editing atmultiple target sites, or multiplex genome editing, have been describedfor both mammalian cells and plants, and can be achieved by expressingone or more sgRNAs to target multiple genome sites within the organism.This has been demonstrated in rice with the use of seven sgRNAs forediting (Ma et al., 2015, Mol Plant, 8, 1274). It is therefore anobjective of this invention to use multiple sgRNAs to direct theinsertion of a specific DNA sequence to multiple sites in the plantgenome using one or more of the previous embodiments of the invention.

Methods for DNA Insertion at the Target Site

The methods for achieving the genome modification are described usingthe CRISPR/Cas9 system although it will be appreciated that othervariations of the CRISPR/Cas systems can also be used including one thatuses guide DNA sequences. The method requires the introduction of thesite-specific nuclease and guide RNA into the nucleus of plant cellsfrom the target crop. These may vary for different crop species or dueto preference or skill set of the crop scientists.

One skilled in the art can produce and introduce proteins or DNA intomany crop types using plant cell protoplasts. Preferably the plantprotoplasts once genome edited can be regenerated into stable fertileplants suitable for crop breeding programs. For example, protoplasttransformation and hence genome editing is useful for modifying thegenomes of Camelina, canola, soybean, corn, rice, wheat, potato,alfalfa, tomato, cotton, barley and many other crops of interest. TheCas9 nuclease enzyme can be combined with the sgRNAs to form protein/RNAparticles which can then be introduced into the plant protoplasts.

Methods for Identifying or Selecting Plant Cells with the TargetedGenome Edits Methods of Plant Transformation

Known transformations methods can be used upregulate one or more genesequences of the invention.

Vectors

Several plant transformation vector options are available, includingthose described in Gene Transfer to Plants, 1995, Potrykus et al., eds.,Springer-Verlag Berlin Heidelberg New York, Transgenic Plants: AProduction System for Industrial and Pharmaceutical Proteins, 1996, Owenet al., eds., John Wiley & Sons Ltd. Eng, and Methods in Plant MolecularBiology: A Laboratory Course Manual, 1995, Maliga et al., eds., ColdSpring Laboratory Press, New York). Plant transformation vectorsgenerally include one or more coding sequences of interest under thetranscriptional control of 5′ and 3′ regulatory sequences, including apromoter, a transcription termination and/or polyadenylation signal, anda selectable or screenable marker gene.

Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA sequence andinclude vectors such as pBIN19. Typical vectors suitable forAgrobacterium transformation include the binary vectors pCIB200 andpCIB2001, as well as the binary vector pCIB 10 and hygromycin selectionderivatives thereof (See, for example, U.S. Pat. No. 5,639,949).

Transformation without the use of Agrobacterium tumefaciens circumventsthe requirement for T-DNA sequences in the chosen transformation vector,and consequently vectors lacking these sequences are utilized inaddition to vectors such as the ones described above which contain T-DNAsequences. The choice of vector for transformation techniques that donot rely on Agrobacterium depends largely on the preferred selection forthe species being transformed. Typical vectors suitable fornon-Agrobacterium transformation include pCIB3064, pSOG 19, and pSOG35(See, for example, U.S. Pat. No. 5,639,949). Alternatively, DNAfragments containing the transgene and the necessary regulatory elementsfor expression of the transgene can be excised from a plasmid anddelivered to the plant cell using microprojectile bombardment-mediatedmethods.

Protocols

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell targeted for transformation. Suitable methods of introducingnucleotide sequences into plant cells and subsequent insertion into theplant genome include microinjection (Crossway et al. (1986)Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediatedtransformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al. WOUS98/01268), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); and McCabe et al. Biotechnology 6:923-926(1988)). Also see Weissinger et al. Ann. Rev. Genet. 22:421-477 (1988);Sanford et al. Particulate Science and Technology 5:27-37 (1987)(onion); Klein et al. Proc. Natl. Acad. Sci. USA 85:4305-4309 (1988)(maize); Klein et al. Biotechnology 6:559-563 (1988) (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize);Klein et al. Plant Physiol. 91:440-444 (1988) (maize); Fromm et al.Biotechnology 8:833-839 (1990) (maize); Hooykaas-Van Slogteren et al.Nature 311:763-764 (1984); Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. Proc. Natl. Acad. Sci. USA 84:5345-5349(1987) (Liliaceae); De Wet et al. in The Experimental Manipulation ofOvule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (1985)(pollen); Kaeppler et al. Plant Cell Reports 9:415-418 (1990) andKaeppler et al. Theor. Appl. Genet. 84:560-566 (1992) (whisker-mediatedtransformation); D'Halluin et al. Plant Cell 4:1495-1505 (1992)(electroporation); Li et al. Plant Cell Reports 12:250-255 (1993) andChristou and Ford Annals of Botany 75:407-413 (1995) (rice); Osjoda etal. Nature Biotechnology 14:745-750 (1996) (maize via Agrobacteriumtumefaciens). References for protoplast transformation and/or gene gun(also known as biolistics) are described in WO 2010/037209. Methods fortransforming plant protoplasts are available including transformationusing polyethylene glycol (PEG), electroporation, and calcium phosphateprecipitation (see for example Potrykus et al., 1985, Mol. Gen. Genet.,199, 183-188; Potrykus et al., 1985, Plant Molecular Biology Reporter,3, 117-128). Methods for plant regeneration from protoplasts have alsobeen described [Evans et al., in Handbook of Plant Cell Culture, Vol 1,(Macmillan Publishing Co., New York, 1983); Vasil, IK in Cell Cultureand Somatic Cell Genetics (Academic, Oro, 1984)].

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation.

The transformed cells are grown into plants in accordance withconventional techniques. See, for example, McCormick et al., 1986, PlantCell Rep. 5: 81-84. These plants may then be grown, and eitherpollinated with the same transformed variety or different varieties, andthe resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure constitutive expression of the desired phenotypiccharacteristic has been achieved.

In planta methods have also been used for transformation of germ cellsin maize (pollen, Wang et al. 2001, Acta Botanica Sin., 43, 275-279;Zhang et al., 2005, Euphytica, 144, 11-22; pistils, Chumakov et al.2006, Russian J. Genetics, 42, 893-897; Mamontova et al. 2010, RussianJ. Genetics, 46, 501-504) and Sorghum (pollen, Wang et al. 2007,Biotechnol. Appl. Biochem., 48, 79-83).

Selection

Following transformation by any one of the methods described above, thefollowing procedures can be used to obtain a transformed plantexpressing the transgenes: select the plant cells that have beentransformed on a selective medium; regenerate the plant cells that havebeen transformed to produce differentiated plants; select transformedplants expressing the DNA construct for introducing the targetedinsertion of the DNA sequence elements producing the desired level ofdesired polypeptide(s) in the desired tissue and cellular location.

The cells that have been transformed may be grown into plants inaccordance with conventional techniques. See, for example, McCormick etal. Plant Cell Reports 5:81-84(1986). These plants may then be grown,and either pollinated with the same transformed variety or differentvarieties, and the resulting hybrid having constitutive expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that constitutive expression of thedesired phenotypic characteristic is stably maintained and inherited andthen seeds harvested to ensure constitutive expression of the desiredphenotypic characteristic has been achieved.

Transgenic plants can be produced using conventional techniques toexpress any genes of interest in plants or plant cells (Methods inMolecular Biology, 2005, vol. 286, Transgenic Plants: Methods andProtocols, Pena L., ed., Humana Press, Inc. Totowa, N.J.; ShyamkumarBarampuram and Zhanyuan J. Zhang, Recent Advances in PlantTransformation, in James A. Birchler (ed.), Plant ChromosomeEngineering: Methods and Protocols, Methods in Molecular Biology, vol.701, Springer Science+Business Media). Typically, gene transfer, ortransformation, is carried out using explants capable of regeneration toproduce complete, fertile plants. Generally, a DNA or an RNA molecule tobe introduced into the organism is part of a transformation vector. Alarge number of such vector systems known in the art may be used, suchas plasmids. The components of the expression system can be modified,e.g., to increase expression of the introduced nucleic acids. Forexample, truncated sequences, nucleotide substitutions or othermodifications may be employed. Expression systems known in the art maybe used to transform virtually any plant cell under suitable conditions.A transgene comprising a DNA molecule encoding a gene of interest ispreferably stably transformed and integrated into the genome of the hostcells. Transformed cells are preferably regenerated into whole fertileplants. Detailed descriptions of transformation techniques are withinthe knowledge of those skilled in the art.

Plant promoters can be selected to control the expression of thetransgene in different plant tissues or organelles for all of whichmethods are known to those skilled in the art (Gasser & Fraley, 1989,Science 244: 1293-1299). In one embodiment, promoters are selected fromthose of eukaryotic or synthetic origin that are known to yield highlevels of expression in plants and algae. In a preferred embodiment,promoters are selected from those that are known to provide high levelsof expression in monocots.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050, the core CaMV 35S promoter (Odell et al.,1985, Nature 313: 810-812), rice actin (McElroy et al., 1990, Plant Cell2: 163-171), ubiquitin (Christensen et al., 1989, Plant Mol. Biol. 12:619-632; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689), pEMU(Last et al., 1991, Theor. Appl. Genet. 81: 581-588), MAS (Velten etal., 1984, EMBO J. 3: 2723-2730), and ALS promoter (U.S. Pat. No.5,659,026). Other constitutive promoters are described in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; and 5,608,142.

“Tissue-preferred” promoters can be used to target gene expressionwithin a particular tissue. Compared to chemically inducible systems,developmentally and spatially regulated stimuli are less dependent onpenetration of external factors into plant cells. Tissue-preferredpromoters include those described by Van Ex et al., 2009, Plant CellRep. 28: 1509-1520; Yamamoto et al., 1997, Plant J. 12: 255-265;Kawamata et al., 1997, Plant Cell Physiol. 38: 792-803; Hansen et al.,1997, Mol. Gen. Genet. 254: 337-343; Russell et al., 199), TransgenicRes. 6: 157-168; Rinehart et al., 1996, Plant Physiol. 112: 1331-1341;Van Camp et al., 1996, Plant Physiol. 112: 525-535; Canevascini et al.,1996, Plant Physiol. 112: 513-524; Yamamoto et al., 1994, Plant CellPhysiol. 35: 773-778; Lam, 1994, Results Probl. Cell Differ. 20:181-196, Orozco et al., 1993, Plant Mol. Biol. 23: 1129-1138; Matsuokaet al., 1993, Proc. Natl. Acad. Sci. USA 90: 9586-9590, andGuevara-Garcia et al., 1993, Plant J. 4: 495-505. Such promoters can bemodified, if necessary, for weak expression.

Any of the described promoters can be used to control the expression ofone or more of the genes of the invention, their homologs and/ororthologs as well as any other genes of interest in a definedspatiotemporal manner.

Expression Cassettes

Nucleic acid sequences intended for expression in transgenic plants arefirst assembled in expression cassettes behind a suitable promoteractive in plants. The expression cassettes may also include any furthersequences required or selected for the expression of the transgene. Suchsequences include, but are not restricted to, transcription terminators,extraneous sequences to enhance expression such as introns, vitalsequences, and sequences intended for the targeting of the gene productto specific organelles and cell compartments. These expression cassettescan then be transferred to the plant transformation vectors describedinfra. The following is a description of various components of typicalexpression cassettes.

A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and the correct polyadenylation ofthe transcripts. Appropriate transcriptional terminators are those thatare known to function in plants and include the CaMV 35S terminator, thetm1 terminator, the nopaline synthase terminator and the pea rbcS E9terminator. These are used in both monocotyledonous and dicotyledonousplants.

Individual plants within a population of transgenic plants that expressa recombinant gene(s) may have different levels of gene expression. Thevariable gene expression is due to multiple factors including multiplecopies of the recombinant gene, chromatin effects, and gene suppression.Accordingly, a phenotype of the transgenic plant may be measured as apercentage of individual plants within a population. The yield of aplant can be measured simply by weighing. The yield of seed from a plantcan also be determined by weighing. The increase in seed weight from aplant can be due to a number of factors, an increase in the number orsize of the seed pods, an increase in the number of seed or an increasein the number of seed per plant. In the laboratory or greenhouse seedyield is usually reported as the weight of seed produced per plant andin a commercial crop production setting yield is usually expressed asweight per acre or weight per hectare.

A recombinant DNA construct including a plant-expressible gene or otherDNA of interest is inserted into the genome of a plant by a suitablemethod. Suitable methods include, for example, Agrobacteriumtumefaciens-mediated DNA transfer, direct DNA transfer,liposome-mediated DNA transfer, electroporation, co-cultivation,diffusion, particle bombardment, microinjection, gene gun, calciumphosphate coprecipitation, viral vectors, nanotube or nanoparticlemediated delivery, and other techniques. Suitable plant transformationvectors include those derived from a Ti plasmid of Agrobacteriumtumefaciens. In addition to plant transformation vectors derived fromthe Ti or root-inducing (Ri) plasmids of Agrobacterium, alternativemethods can be used to insert DNA constructs into plant cells. Atransgenic plant can be produced by selection of transformed seeds or byselection of transformed plant cells and subsequent regeneration.

In one embodiment, the transgenic plants are grown (e.g., on soil) andharvested. In one embodiment, above ground tissue is harvestedseparately from below ground tissue. Suitable above ground tissuesinclude shoots, stems, leaves, flowers, grain, and seed. Exemplary belowground tissues include roots and root hairs. In one embodiment, wholeplants are harvested and the above ground tissue is subsequentlyseparated from the below ground tissue.

Genetic constructs may encode a selectable marker to enable selection oftransformation events. There are many methods that have been describedfor the selection of transformed plants [for review see (Miki et al.,Journal of Biotechnology, 2004, 107, 193-232) and referencesincorporated within]. Selectable marker genes that have been usedextensively in plants include the neomycin phosphotransferase gene nptII(U.S. Pat. Nos. 5,034,322, 5,530,196), hygromycin resistance gene (U.S.Pat. No. 5,668,298, Waldron et al., (1985), Plant Mol Biol, 5:103-108;Zhijian et al., (1995), Plant Sci, 108:219-227), the bar gene encodingresistance to phosphinothricin (U.S. Pat. No. 5,276,268), the expressionof aminoglycoside 3″-adenyltransferase (aadA) to confer spectinomycinresistance (U.S. Pat. No. 5,073,675), the use of inhibition resistant5-enolpyruvyl-3-phosphoshikimate synthetase (U.S. Pat. No. 4,535,060)and methods for producing glyphosate tolerant plants (U.S. Pat. Nos.5,463,175; 7,045,684). Other suitable selectable markers include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella et al., (1983), EMBO J, 2:987-992), methotrexate(Herrera Estrella et al., (1983), Nature, 303:209-213; Meijer et al,(1991), Plant Mol Biol, 16:807-820); streptomycin (Jones et al., (1987),Mol Gen Genet, 210:86-91); bleomycin (Hille et al., (1990), Plant MolBiol, 7:171-176); sulfonamide (Guerineau et al., (1990), Plant Mol Biol,15:127-136); bromoxynil (Stalker et al., (1988), Science, 242:419-423);glyphosate (Shaw et al., (1986), Science, 233:478-481); phosphinothricin(DeBlock et al., (1987), EMBO J, 6:2513-2518).

Methods of plant selection that do not use antibiotics or herbicides asa selective agent have been previously described and include expressionof glucosamine-6-phosphate deaminase to inactive glucosamine in plantselection medium (U.S. Pat. No. 6,444,878) and a positive/negativesystem that utilizes D-amino acids (Erikson et al., Nat Biotechnol,2004, 22, 455-8). European Patent Publication No. EP 0 530 129 A1describes a positive selection system which enables the transformedplants to outgrow the non-transformed lines by expressing a transgeneencoding an enzyme that activates an inactive compound added to thegrowth media. U.S. Pat. No. 5,767,378 describes the use of mannose orxylose for the positive selection of transgenic plants.

Methods for positive selection using sorbitol dehydrogenase to convertsorbitol to fructose for plant growth have also been described (WO2010/102293). Screenable marker genes include the beta-glucuronidasegene (Jefferson et al., 1987, EMBO J. 6: 3901-3907; U.S. Pat. No.5,268,463) and native or modified green fluorescent protein gene (Cubittet al., 1995, Trends Biochem. Sci. 20: 448-455; Pan et al., 1996, PlantPhysiol. 112: 893-900).

Transformation events can also be selected through visualization offluorescent proteins such as the fluorescent proteins from thenonbioluminescent Anthozoa species which include DsRed, a redfluorescent protein from the Discosoma genus of coral (Matz et al.(1999), Nat Biotechnol 17: 969-73). An improved version of the DsRedprotein has been developed (Bevis and Glick (2002), Nat Biotech 20:83-87) for reducing aggregation of the protein.

Visual selection can also be performed with the yellow fluorescentproteins (YFP) including the variant with accelerated maturation of thesignal (Nagai, T. et al. (2002), Nat Biotech 20: 87-90), the bluefluorescent protein, the cyan fluorescent protein, and the greenfluorescent protein (Sheen et al. (1995), Plant J 8: 777-84; Davis andVierstra (1998), Plant Molecular Biology 36: 521-528). A summary offluorescent proteins can be found in Tzfira et al. (Tzfira et al.(2005), Plant Molecular Biology 57: 503-516) and Verkhusha and Lukyanov(Verkhusha, V. V. and K. A. Lukyanov (2004), Nat Biotech 22: 289-296)whose references are incorporated in entirety. Improved versions of manyof the fluorescent proteins have been made for various applications.Based on the disclosure herein, it will be apparent to a person of skillin the art how to use of the improved versions of these proteins orcombinations of these proteins for selection of transformants.

The plants modified for enhanced performance by increasing theexpression of the transcription factor genes or transcription factorgene combinations may be combined or stacked with input traits bycrossing or plant breeding. Useful input traits include herbicideresistance and insect tolerance, for example a plant that is tolerant tothe herbicide glyphosate and that produces the Bacillus thuringiensis(BT) toxin. Glyphosate is a herbicide that prevents the production ofaromatic amino acids in plants by inhibiting the enzyme5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase). Theoverexpression of EPSP synthase in a crop of interest allows theapplication of glyphosate as a weed killer without killing the modifiedplant (Suh, et al., J. M Plant Mol. Biol. 1993, 22, 195-205). BT toxinis a protein that is lethal to many insects providing the plant thatproduces it protection against pests (Barton, et al. Plant Physiol.1987, 85, 1103-1109). Other useful herbicide tolerance traits includebut are not limited to tolerance to Dicamba by expression of the dicambamonoxygenase gene (Behrens et al, 2007, Science, 316, 1185), toleranceto 2,4-D and 2,4-D choline by expression of a bacterial aad-1 gene thatencodes for an aryloxyalkanoate dioxygenase enzyme (Wright et al.,Proceedings of the National Academy of Sciences, 2010, 107, 20240),glufosinate tolerance by expression of the bialophos resistance gene(bar) or the pat gene encoding the enzyme phosphinotricin acetyltransferase (Droge et al., Planta, 1992, 187, 142), as well as genesencoding a modified 4-hydroxyphenylpyruvate dioxygenase (HPPD) thatprovides tolerance to the herbicides mesotrione, isoxaflutole, andtembotrione (Siehl et al., Plant Physiol, 2014, 166, 1162). The plantsmodified for enhanced yield by reducing the expression of thetranscription factor genes or transcription factor gene combinations maybe combined or stacked with other genes which improve plant performance.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this present invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.

All patents, publications and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES Example 1. Identification of Maize Orthologs to SwitchgrassTranscription Factors

Over expression of the switchgrass transcription factors STR1 (SEQ IDNOS: 1 and 2), STIF1 (SEQ ID NOS: 11 and 12), and BMY1 (SEQ ID NOS: 21and 22) in switchgrass have been previously been shown to increasebiomass yield, photosynthetic parameters, and the content ofphotosynthetic pigments, soluble sugars, and starch and a maize sequencebased ortholog for each switchgrass gene has been identified(WO2014100289). The switchgrass transcription factors were originallyidentified from a rice transcriptome regulatory association network(WO2014100289). Improvements to whole-genome datasets has more recentlyallowed the identification of additional orthologs to STR1, STIF1, andBMY1 in maize (TABLE 1 and TABLE 2) as well as homologs to STR1, STIF1,and BMY1 in switchgrass (TABLE 2). Manipulation of expression of thesegenes, through transgenic or genome editing approaches, can be used toincrease yield in maize.

To identify additional maize transcription factor genes, the switchgrassSTR1 (SEQ ID NO: 2), STIF1 (SEQ ID NO: 12), and BMY1 (SEQ ID NO: 22)proteins were used. The switchgrass amino acid sequence of eachtranscription factor was blasted against the maize proteome(Phytozome-Ensemlb-18). The hits were ranked in order of the alignmentscore. Next each maize amino acid sequence was aligned to theswitchgrass sequence using the alignment feature of the Vector NTIsoftware (Invitrogen) to determine the percent identity between theswitchgrass and maize orthologs. A summary of the gene and proteinsequences of the maize orthologs is shown in TABLE 1. The CLUSTALO(1.2.4) multiple sequence alignment tool was used to align eachswitchgrass transcription factor to its switchgrass homologs and itsmaize orthologs and these alignments are shown in FIG. 1 (STR1), FIG. 2(STIF1), and FIG. 3 (BMY1). Key characteristics shared among variousmaize orthologs of STR1 from switchgrass (SEQ ID NO:2) and the STR1switchgrass protein itself include a tryptophan at position 24, anarginine at position 38, a region of high identity/similarity betweenpositions 102 and 156, a proline at position 212, a glutamine atposition 303, a leucine at position 311, and a proline at position 318,all with numbering of positions relative to STR1 of switchgrass of SEQID NO: 2. Key characteristics shared among various maize orthologs ofSTIF1 from switchgrass (SEQ ID NO: 12) and the STIF1 switchgrass proteinitself include a tyrosine at position 4, an alanine at position 25, ahistidine at position 37, a region of high identity/similarity betweenpositions 73 and 129, a threonine at position 136, a glycine at position146, a proline at position 167, a leucine at position 169, a tyrosine atposition 172, and an alanine at position 173, all with numbering ofpositions relative to STIF1 of switchgrass of SEQ ID NO: 12. Keycharacteristics shared among various maize orthologs of BMY1 fromswitchgrass (SEQ ID NO: 22) and the BMY1 switchgrass protein itselfinclude a methionine at position 1, a glutamic acid at position 7, aserine at position 8, a glycine at position 9, a region of highidentity/similarity between positions 17 and 114, a serine at position137, a glycine at position 149, and a tyrosine at position 151, all withnumbering of positions relative to BMY1 of switchgrass of SEQ ID NO: 22.

TABLE 1 Maize orthologs and homologs to the switchgrass transcriptionfactors STR1, STIF1, and BMY1 Switchgrass TF¹ Maize Ortholog 1 MaizeOrtholog 2 Maize Ortholog 3 Maize Ortholog 4 Maize Ortholog 5 STR1 geneGRMZM2G018398 GRMZM2G110333 GRMZM2G171179 GRMZM2G018984 GRMZM2G142179(Pavir.Ba00410) SEQ ID NO: 3 SEQ ID NO: 5 SEQ ID NO: 7 SEQ ID NO: 9 SEQID NO: 31 SEQ ID NO: 1 STR1 protein SEQ ID NO: 4 SEQ ID NO: 6 SEQ ID NO:8 SEQ ID NO: 10 SEQ ID NO: 32 SEQ ID NO: 2 (33.2% identity to (33.1%identity to (35.1% identity to (33.7% identity to (22.4% identity toswitchgrass STR1) switchgrass STR1) switchgrass STR1) switchgrass STR1)switchgrass STR1) STIF1 gene GRMZM2G016434 GRMZM2G087059 GRMZM2G425798GRMZM2G309731 (Pavir.Aa02595) SEQ ID NO: 13 SEQ ID NO: 15 SEQ ID NO: 17SEQ ID NO: 19 SEQ ID NO: 11 STIF1 protein SEQ ID NO: 14 SEQ ID NO: 16SEQ ID NO: 18 SEQ ID NO: 20 SEQ ID NO: 12 (34.7% identity to (24.4%identity to (26.7% identity to (30.2% identity to switchgrass STIF1)switchgrass STIF1) switchgrass STIF1) switchgrass STIF1) BMY1 geneGRMZM2G384528 GRMZM2G180947 GRMZM2G064426 GRMZM5G804893 (Pavir.J05081)SEQ ID NO: 23 SEQ ID NO: 25 SEQ ID NO: 27 SEQ ID NO: 29 SEQ ID NO: 21BMY1 protein SEQ ID NO: 24 SEQ ID NO: 26 SEQ ID NO: 28 SEQ ID NO: 30 SEQID NO: 22 (78.6% identity to (76.3% identity to (45.0% identity to(45.4% identity to switchgrass BMY1) switchgrass BMY1) switchgrass BMY1)switchgrass BMY1) ¹gene ID from Phytozome v12.0

Since switchgrass is a tetraploid, available sequence data forswitchgrass (Panicum virgatum genotype AP13) available on Phytozome(version 12.1.6) was used to identify additional switchgrasstranscription factors with homology to the switchgrass protein sequencesof STR1 (SEQ ID NO: 2), STIF1 (SEQ ID NO: 12), and BMY1 (SEQ ID NO: 22).The switchgrass amino acid sequence of each TF was blasted against theswitchgrass proteome (Phytozome version 12.1.6). The hits were ranked inorder of the alignment score and the top hits are shown in the firstcolumn in TABLE 2. These new switchgrass proteins were used to identifynew maize orthologs as follows: the switchgrass amino acid sequence ofeach TF was blasted against the maize proteome (Phytozome-Ensemlb-18)and the hits were ranked in order of the alignment score. Most of themaize orthologs obtained from this process were the same orthologspreviously listed in TABLE 1, however three new orthologs including theGRMZM2G457562 protein (SEQ ID NO: 41), the GRMZM2G100727 protein (SEQ IDNO: 43), and the GRMZM2G303465 protein (SEQ ID NO: 48) were identified.

TABLE 2 Switchgrass orthologs to the switchgrass transcription factorsSTR1, STIF1, and BMY1 and their maize orthologs and homologs. Maizeortholog 1 Maize ortholog 2 Maize ortholog 3 Switchgrass proteins¹ withhomology to STR1 Pavir.Bb03337 protein GRMZM2G018398 gene GRMZM2G110333GRMZM2G171179 gene (SEQ ID NO: 33) (SEQ ID NO: 3) gene (SEQ ID NO: 5)(SEQ ID NO: 7) GRMZM2G018398 protein GRMZM2G110333 GRMZM2G171179 (SEQ IDNO: 4) protein (SEQ ID NO: 6) protein (SEQ ID NO: 8) Pavir.J04875protein GRMZM2G018398 gene GRMZM2G110333 GRMZM2G171179 gene (SEQ ID NO:51) (SEQ ID NO: 3) gene (SEQ ID NO: 5) (SEQ ID NO: 7) GRMZM2G018398protein GRMZM2G110333 GRMZM2G171179 (SEQ ID NO: 4) protein (SEQ ID NO:6) protein (SEQ ID NO: 8) Pavir.Aa00281 protein GRMZM2G018398 geneGRMZM2G110333 GRMZM2G171179 gene (SEQ ID NO: 35) (SEQ ID NO: 3) gene(SEQ ID NO: 5) (SEQ ID NO: 7) GRMZM2G018398 protein GRMZM2G110333GRMZM2G171179 (SEQ ID NO: 4) protein (SEQ ID NO: 6) protein (SEQ ID NO:8) Pavir.Ib00526 protein GRMZM2G018984 gene GRMZM2G018398 GRMZM2G171179gene (SEQ ID NO: 36) (SEQ ID NO: 9) gene (SEQ ID NO: 3) (SEQ ID NO: 7)GRMZM2G018984 protein GRMZM2G018398 GRMZM2G171179 (SEQ ID NO: 10)protein (SEQ ID NO: 4) protein (SEQ ID NO: 8) Switchgrass proteins withhomology to STIF1 Pavir.Gb01735.1 protein GRMZM2G425798 geneGRMZM2G087059 GRMZM2G016434 gene (SEQ ID NO: 38) (SEQ ID NO: 17) gene(SEQ ID NO: 15) (SEQ ID NO: 13) GRMZM2G425798 protein GRMZM2G087059GRMZM2G016434 (SEQ ID NO: 18) protein (SEQ ID NO: 16) protein (SEQ IDNO: 14) Pavir.J04335.1 protein GRMZM2G087059 gene GRMZM2G457562GRMZM2G100727 gene (SEQ ID NO: 15) gene (SEQ ID NO: 40) (SEQ ID NO: 42)(SEQ ID NO: 39) GRMZM2G087059 protein GRMZM2G457562 GRMZM2G100727 (SEQID NO: 16) protein (SEQ ID NO: 41) protein (SEQ ID NO: 43) Switchgrassproteins with homology to BMY1 Pavir.Ba00451 protein GRMZM2G180947 geneGRMZM2G384528 GRMZM2G064426 gene (SEQ ID NO: 44) (SEQ ID NO: 25) gene(SEQ ID NO: 23) (SEQ ID NO: 27) GRMZM2G180947 protein GRMZM2G384528GRMZM2G064426 (SEQ ID NO: 26) protein (SEQ ID NO: 24) protein (SEQ IDNO: 28) Pavir.Ib01924 protein GRMZM2G180947 gene GRMZM2G384528GRMZM2G064426 gene (SEQ ID NO: 45) (SEQ ID NO: 25) gene (SEQ ID NO: 23)(SEQ ID NO: 27) GRMZM2G180947 protein GRMZM2G384528 GRMZM2G064426 (SEQID NO: 26) protein (SEQ ID NO: 24) protein (SEQ ID NO: 28) Pavir.J02009protein GRMZM2G064426 gene GRMZM5G804893 GRMZM2G303465 gene (SEQ ID NO:46) (SEQ ID NO: 27) gene (SEQ ID NO: 29) (SEQ ID NO: 47) GRMZM2G064426protein GRMZM5G804893 GRMZM2G303465 (SEQ ID NO: 28) protein (SEQ ID NO:30) protein (SEQ ID NO: 48) Pavir.Eb03638 protein GRMZM2G303465 geneGRMZM5G804893 GRMZM2G064426 gene (SEQ ID NO: 49) (SEQ ID NO: 47) gene(SEQ ID NO: 29) (SEQ ID NO: 27) GRMZM2G303465 protein GRMZM5G804893GRMZM2G064426 (SEQ ID NO: 48) protein (SEQ ID NO: 30) protein (SEQ IDNO: 28) Pavir.J02756 protein GRMZM2G064426 gene GRMZM5G804893GRMZM2G303465 gene (SEQ ID NO: 50) (SEQ ID NO: 27) gene (SEQ ID NO: 29)(SEQ ID NO: 47) GRMZM2G064426 protein GRMZM5G804893 GRMZM2G303465 (SEQID NO: 28) protein (SEQ ID NO: 30) protein (SEQ ID NO: 48) ¹protein IDfrom Phytozome v12.1.6

Example 2. Expression Patterns of Select Transcription Factors in Corn

The in silico expression pattern of select maize orthologs to STR1(GRMZM2G110333, SEQ ID NO: 5), STIF1 (GRMZM2G016434, SEQ 13) and BMY1(GRMZM2G384528, SEQ ID NO: 23) were examined using the maize ElectronicFluorescent Pictograph browser (Li, L. et al., Nat Genet, 42 (2010)1060-1067) (FIG. 4A-C). Surprisingly, the genes for GRMZM2G110333 (SEQID NO: 5) and GRMZM2G384528 (SEQ ID NO: 23) were found to have thehighest level of expression in developing and whole seed tissue.GRMZM2G016434, (SEQ 13) also had expression in developing seed and wholeseed with the highest levels in the 1^(st) leaf and sheath.

The expression of these genes was also experimentally determined byRT-PCR analysis. Maize plants (inbred line B73 obtained from The NorthCentral Regional Plant Introduction Station, Iowa State University) weregrown in a greenhouse and tissue at different developmental stages washarvested. The levels of amplification products (FIG. 4D) were measuredin 50 ng of total RNA using One Step RT-PCR Kit (Qiagen, Valencia,Calif., USA) as described previously (Somleva, et al., BMC Biotechnol.,14 (2014) 79) using the following pairs of primers:5′CGTGTTTGGCTTGGTACTTTC3′ and 5′GGAAGTGATGTCTGGTGTCTT3′ forGRMZM2G110333 (SEQ ID NO: 5); TACTCTGACCACGACGATGA andGCAACAACGGAGCTGATACT for GRMZM2G016434 (SEQ ID NO: 13); and5′GTCGGAGTTCATCTCCTTCATC3′ and 5′ TCATCATGATCATACCGCTTCC3′ forGRMZM2G384528 (SEQ ID NO: 23). Amplification conditions were as follows:50° C. for 30 min; 95° C. for 15 min; 94° C. for 1 min, 55° C. for 30sec, 72° C. for 1 min (30 cycles); extension at 72° C. for 15 min. Ourexperimental examination of the expression pattern of the genesconfirmed that GRMZM2G110333 (SEQ ID NO: 5), GRMZM2G016434 (SEQ 13) andGRMZM2G384528 (SEQ ID NO: 23) were expressed in leaves important forproviding photoassimilates during seed formation, as well as in thepre-pollination cob and the whole seed 12 days after pollination (FIG.4D). This suggests a role for the maize transcription factor genes inregulating processes during seed formation that impact seed yield.

Example 3. Overexpression of Transcription Factors in Corn

Expression cassettes for the maize orthologs of the switchgrasstranscription factor proteins STR1 (SEQ ID NO: 2), STIF1 (SEQ ID NO:12), and BMY1 (SEQ ID NO: 22) can be constructed using a variety ofdifferent promoters for expression. Candidate constitutive and seedspecific promoters are listed in TABLE 3 and TABLE 4, however thoseskilled in the art will understand that other promoters can be selectedfor expression.

TABLE 3 Example promoters for expression in maize Maize gene ID¹Promoter Expression (SEQ ID #)² Hsp70 Constitutive GRMZM2G310431 (SEQ IDNO: 57) Chlorophyll A/B Light inducible, AC207722.2_FG009 BindingProtein expressed in maize (SEQ ID NO: 58) (Cab-m5) mesophyll andGRMZM2G351977 bundlesheath cells (SEQ ID NO: 59) Pyruvate ConstitutiveGRMZM2G306345 phosphate (SEQ ID NO: 60) dikinase (PPDK) ActinConstitutive GRMZM2G047055 (SEQ ID NO: 61) ADP-glucose Seed specificGRMZM2G429899 pyrophos- (SEQ ID NO: 62) phorylase (AGPase) β- Seedspecific GRMZM2G139300 fructofuranosidase (SEQ ID NO: 63) insolubleisoenzyme 1 (CIN1) Maize MADS box Seed specific GRMZM2G160687 promoter(SEQ ID NO: 56) Maize trpA Seed specific GRMZM5G841619 promoter (SEQ IDNO: 74) ¹Gene ID on Phytozyme v. 12.1.6; ²Promoter region includes thepredicted 5′UTR of the gene and 1200 bp of sequence upstream of the5′UTR in Phytozyme v. 12.1.6

In some instances, it may be advantageous to create a hybrid promotercontaining a promoter sequence and an intron. These promoters candeliver higher levels of Mable expression. Examples of such hybridpromoters are listed in TABLE 4.

TABLE 4 Hybrid promoter replacement cassettes Promoter Expression Hybridmaize Cab-m5 Light inducible, expressed in maize SEQ ID NO: 64promoter/maize hsp70 mesophyll and bundlesheath cells intron Maizeubiquitin Constitutive (maize ubiquitin promoter SEQ ID NO: 65promoter/maize and intron sequence listed in Genbank ubiquitin intronKT962835) Maize ubiquitin Constitutive (maize promoter and intron SEQ IDNO: 70 promoter/maize sequence with 99% identity to sequence ubiquitinintron in Genbank KT985051.1) Maize ubiquitin Constitutive promoter/adh1intron 1 Rice actin Constitutive promoter/actin intron 1 Maize H2B(histone) Constitutive promoter/ubiquitin intron 1

Expression cassettes for maize gene GRMZM2G384528 (SEQ ID NO: 23), oneof the maize orthologs for the switchgrass BMY1 transcription factorgene (SEQ ID NO: 21) were designed using different promoters to driveexpression of the transgenes. YTEN26 (FIG. 5A, SEQ ID NO: 66) isexpressed from the hybrid maize cab-m5/maize hsp70 intron promoter (SEQID NO: 64) and is flanked by maize hsp70 terminator. The cab-m5 promoterhas been previously shown to be light inducible and expressed in bothmesophyll and bundlesheath cells of maize, with some preference formesophyll (Sheen et al., P. Natl. Acad. Sci. USA, 1986, 83, 7811).YTEN27 (FIG. 5B, SEQ ID NO: 67) is expressed from the maize MADS-boxpromoter (SEQ ID NO: 56) and is flanked at the 3′ end by the hsp70terminator. YTEN28 (FIG. 5C, SEQ ID NO: 68) is expressed from the maizetrpA promoter (SEQ ID NO: 74) and is flanked at the 3′ end by the hsp70terminator. YTEN29 (FIG. 5D, SEQ ID NO: 69) is expressed from the maizeubiquitin promoter with the maize ubiquitin intron 1 (SEQ ID NO: 65) andis flanked at the 3′ end by the hsp70 terminator.

Maize: Methods for maize transformation are routine and well known inthe art and have recently been reviewed by Que et al., (2014), Frontiersin Plant Science 5, article 379, pp 1-19.

Protoplast transformation: Protoplast transformation methods useful forpracticing the invention are well known to those skilled in the art.Such procedures include for example the transformation of maizeprotoplasts as described by Rhodes and Gray (Rhodes, C. A. and D. W.Gray, Transformation and regeneration of maize protoplasts, in PlantTissue Culture Manual: Supplement 7, K. Lindsey, Editor. 1997, SpringerNetherlands: Dordrecht. p. 353-365).

Agrobacterium-mediated transformation: For transformation of maize,fragments from YTEN26, YTEN 27, YTEN28, or YTEN29 can be inserted into abinary vector that also contains an expression cassette for a selectablemarker. For example the bar gene imparting the transgenic plants withresistance to bialophos can be used for selection. The binary vector istransformed into an Agrobacterium tumefaciens strain, such as A.tumefaciens strain EHA101.

Agrobacterium-mediated transformation of maize can be performedfollowing a previously described procedure (Frame et al., 2006,Agrobacterium Protocols Wang K., ed., Vol. 1, pp 185-199, Humana Press)as follows.

Plant Material: Plants grown in a greenhouse are used as an explantsource. Ears are harvested 9-13 days after pollination and surfacesterilized with 80% ethanol.

Explant Isolation, Infection and Co-Cultivation: Immature zygoticembryos (1.2-2.0 mm) are aseptically dissected from individual kernelsand incubated in an A. tumefaciens strain EHA101 culture containing thetransformation vector of interest for genome editing (grown in 5 ml N6medium supplemented with 100 μM acetosyringone for stimulation of thebacterial vir genes for 2-5 h prior to transformation) at roomtemperature for 5 min. The infected embryos are transferred scutellumside up on to a co-cultivation medium (N6 agar-solidified mediumcontaining 300 mg/l cysteine, 5 μM silver nitrate and 100 μMacetosyringone) and incubated at 20° C., in the dark for 3 d. Embryosare transferred to N6 resting medium containing 100 mg/l cefotaxime, 100mg/l vancomycin and 5 μM silver nitrate and incubated at 28° C., in thedark for 7 d.

Callus Selection: All embryos are transferred on to the first selectionmedium (the resting medium described above supplemented with 1.5 mg/lbialaphos) and incubated at 28° C. in the dark for 2 weeks followed bysubculture on a selection medium containing 3 mg/l bialaphos.Proliferating pieces of callus are propagated and maintained bysubculture on the same medium every 2 weeks.

Plant Regeneration and Selection: Bialaphos-resistant embryogenic calluslines are transferred on to regeneration medium I (MS basal mediumsupplemented with 60 g/l sucrose, 1.5 mg/l bialaphos and 100 mg/lcefotaxime and solidified with 3 g/l Gelrite) and incubated at 25° C. inthe dark for 2 to 3 weeks. Mature embryos formed during this period aretransferred on to regeneration medium II (the same as regenerationmedium I with 3 mg/l bialaphos) for germination in the light (25° C.,80-100 μmol/m²/s light intensity, 16/8-h photoperiod). Regeneratedplants are ready for transfer to soil within 10-14 days. Plants aregrown in the greenhouse to maturity and T1 seeds are isolated.

The copy number of the transgene insert is determined, through methodssuch as Southern blotting or digital PCR, and lines are selected tobring forward for further analysis. Overexpression of the transcriptionfactors is determined by RT-PCR and/or Western blotting techniques andplants with the desired level of expression are selected. Homozygouslines are generated. The yield seed of homozygous lines is compared tocontrol lines.

Transformation using nanotubes or nanoparticles: Nanoparticles ornanotubes capable of delivering biomolecules to plants can also be usedto practice the invention (for review see Cunningham, 2018, TrendsBiotechnol., 36, 882).

Stress experiments, where transgenic plants and their control plants aresubjected to drought, nitrogen deficiency, flooding, heat stress, coldstress, and/or salinity, can also be performed to identify transcriptionfactors that provide stress tolerance.

Example 4. Modulating Expression of Transcription Factors UsingCRISPR/Cas Genome Editing Mediated Promoter Replacement

Methods for targeted mutagenesis, precise gene editing, andsite-specific gene insertion in maize using Cas9 and guide RNA haverecently been published (Svitashev, S., Young, J. K., Schwartz, C., Gao,H., Falco, S. C. and Cigan, A. M. 2015. Plant Physiology 169, 931-945).The expression of a transcription factor can be modulated by replacingthe endogenous promoter in front of the transcription factor with a newpromoter that is expressed at a higher or lower level, is expressed at adifferent developmental stage, and/or has a different tissuespecificity. To modulate expression of the maize orthologs of theswitchgrass transcription factors STR1 (SEQ ID NO: 2), STIF1 (SEQ ID NO:12), and BMY1 (SEQ ID NO: 22), CRISPR/Cas9 mediated promoter replacementcan be used.

Promoter replacement requires the delivery of three elements to theplant, the sgRNAs to target the insertion site, the promoter cassettefor insertion that is flanked by regions homologous to the genomeinsertion site, and the Cas nuclease enzyme. The flanking regions withhomology to the genome insertion site enable incorporation of thepromoter cassette through the plants endogenous homology directed repairmechanism. Delivery of the necessary genetic elements to enable promoterreplacement can be achieved in multiple ways: by introducing a complexof the Cas9 enzyme, the synthesized sgRNAs, and the promoter cassette tobe inserted (called ribonucleoprotein complexes, or RNPs) (FIG. 7C)directly to protoplasts (Woo et al., Nature Biotechnology, 2015, 33,1162-1164); by transfection of protoplasts either stably or transientlywith a genetic construct(s) containing expression cassettes for DNAencoding the sgRNA(s) and the Cas9 enzyme, mixed with a DNA fragmentcontaining the promoter to be inserted (FIG. 7B); through particlebombardment of the plant or plant tissues with a genetic construct(s)with expression cassettes for DNA encoding the sgRNA(s) and the Cas9enzyme, mixed with a DNA fragment containing the promoter to be inserted(FIG. 7B); or through Agrobacterium-mediated transformation of the plantor plant tissues using a binary construct(s) with expression cassettesfor DNA encoding the sgRNA(s), the Cas9 enzyme, and the promoter DNAfragment to be inserted (FIG. 7B). For Agrobacterium-mediatedtransformation, it is advantageous to have the promoter DNA fragment tobe inserted flanked by sgRNA binding sites with adjacent PAM sequences,so that Cas9 expression can release the promoter fragment from thevector as it enters the plant, or alternatively can release the promoterfragment from the T-DNA that is stably incorporated into the plantgenome.

An advantage of RNPs, as well as the protoplast or particle bombardmentmethods, with only transient expression of the expression cassettesencoding the Cas9 enzyme and the sgRNAs, is that DNA does not stablyintegrate into the genome and thus does not need to be removed throughsegregation to produce a plant containing only the edit. For stabletransformation methods, segregation of the unwanted DNA encoding theCRISPR editing machinery must be removed after the edit is obtained byconventional breeding methods. The design of each genetic component toachieve promoter replacement is described below.

Design of single guide RNAs (sgRNAs): The region around the promoter tobe replaced in the genome is scanned for protospacer adjacent motif(PAM) sites, sites necessary for Cas9 to bind and cleave the targetsequence. These PAM sites flank the 3′ region of the double stranded DNAcut site for the Cas9 enzyme (FIG. 6C).

From the ˜20 nucleotides of DNA sequence upstream from the PAM site, thesequence of the complementary “guide” can be obtained (FIG. 6C). Togenerate the functional sgRNA sequence, the sequence of the “guide” iscombined with the sequence of a guide RNA scaffold (FIG. 6B). Guide RNAscaffolds have been previously described by other researchers (see forexample Mali et al. 2013, Science, 339, pp. 823-826; Li et al. 2013,Nature biotechnology, 31, pp. 688-691; Konermann et al., 2015, Nature,517, p. 583; Jiang et al., 2013, Nucleic acids research, 41, pp.e188-e188) and are well known in the art. The double stranded DNAsequence (FIG. 6A) required to generate the functional sgRNA (FIG. 6B)can be determined from the sequence of the sgRNA and used in a genetictransformation construct.

Ideally, the sequence of the DNA encoding the “guide” (FIG. 6A) isidentical to the genomic DNA sequence, or “guide target sequence”, thatis base paired to the sgRNA (FIG. 6C). In practice, some mismatchesbetween the sequence of the DNA encoding the guide (FIG. 6A) and thegenomic DNA sequence can be tolerated and still result in doublestranded cleavage by Cas9.

DNA encoding the guides (FIG. 6A) necessary to generate sgRNAs (FIG. 6B)to excise promoter regions from the maize orthologs of the switchgrasstranscription factors STR1 (SEQ ID NO: 1), STIF1 (SEQ ID NO: 11), andBMY1 (SEQ ID NO: 21) were designed by identifying promoter regionsupstream of the start codon and the 5′UTR of each ortholog. Thistypically contained sequence before the ATG of the coding sequence (CDS)that included 1000-1200 bp of sequence upstream of the 5′UTR (TABLE 5,FIG. 7A). Verification that the specific sequence contains a predictedpromoter can be performed using the RegSite Plant DB from Softberry Inc.(website:softberry.com/berry.phtml?topic=index&group=programs&subgroup=promoter)or similar programs.

DNA sequences encoding the guide portion of the sgRNA for three sgRNAsare shown in TABLE 5. These DNA sequences are ˜20 nucleotides in lengthand span different regions of the upstream promoter (FIG. 7A). Whenfused to DNA encoding the guide RNA scaffold (gRNA Sc) (FIG. 6A), thetranscribed product is a functional sgRNA (FIG. 6B) that has all theelements to bind with the complementary target genomic DNA that liesadjacent to a PAM sequence (FIG. 6C) and to interact with the CASenzyme. The DNA sequences encoding the guide portion of sgRNA shown inTABLE 5 were designed to be components of three sgRNA sequences totarget various regions of the endogenous maize promoter of atranscription factor gene. The use of two sgRNAs can allow for targetedexcision of a region of the endogenous promoter, which can be the corebase elements of the promoter, for example the −10 and −35 regions, orcan include a large fragment encompassing the entire promoter region anduntranslated regions. The use of a single sgRNA promotes site specificcleavage of DNA within the region of the endogenous promoter. Thepositions of the upstream promoter region that are targeted by the sgRNAsequences are outlined in FIG. 7A. DNA sequences encoding the guideportion of sgRNA were designed following the SpCas9 guide RNAarchitectures (equivalent to 20 nucleotides of the target genomic DNAthat is adjacent to a PAM sequence of NGG) using a web-based guide RNAdesign tool, CRISPOR, on the TEFOR website. A number of other web-basedtools can also be used for guide sequence selection and analysis, suchas CRISPRdirect and CRISPR-P 2.0 (Ding et al., 2016, Frontiers in PlantScience, 7, 703; Naito et al., 2015, Bioinformatics, 31, 1120; Liu etal., 2017, Molecular Plant, 10, 530). Based on the disclosure herein, itwill be apparent to a person of skill in the art that different sgRNAsto target different regions of the endogenous promoter for promoterinsertion or replacement can also be used to modulate the expression ofthe maize orthologs of the switchgrass transcription factors STR1 (SEQID NO: 1), STIF1 (SEQ ID NO: 11), and BMY1 (SEQ ID NO: 21). Threedifferent DNA sequences encoding guide portions of three different sgRNAare designated as Guide 1, Guide 2, and Guide 3 in TABLE 5. When thesesequences are transcribed as part of a DNA molecule containing thesequence encoding the RNA scaffold, they produce a functional sgRNA thattargets the regions around the promoter and 5′UTR region for each maizetranscription factor listed in TABLE 5. Similar DNA sequences encodingthe guide portion of sgRNAs can be designed for all of the maize geneslisted in TABLE 1 and TABLE 2 using the upstream promoter sequencesdescribed in TABLE 5 and TABLE 6.

TABLE 5Guide target sequences for Cas9 mediated excision of promoters of transcription factor genes in cornLength of upstream Guide #1² Guide #2 Guide #3 region from Guide GuideGuide Maize CDS used sequence sequence sequence Gene Gene locus namefor analysis¹ Strand³ (5' to 3') PAM⁴ Strand (5' to 3') PAM Strand(5' to 3') PAM STR1 GRMZM2G110333 1113 - GTAAAC GGG + TAGAGTA TGG +CAATTA AGG ortholog (SEQ ID NO: 5) (SEQ ID AAATCG GAATTTC CGAGTA NO: 52)GTGCTTG AAATGG TTAAAT C (SEQ ID (nt 752-771 GC (nt NO: 90) of SEQ ID462-481 NO: 52) of SEQ ID NO: 52) STR1 GRMZM2G142179 1452 + CATACCAGGG + CCGGCTC AGG - AGTAAT GGG ortholog (SEQ ID NO: 31) (SEQ ID AAGCGTCAGCTGTC TTCGGG NO: 53) GGAAGA ATTTAC ATTCAC (nt 1368- (nt 748-767GA (SEQ 1387 of of SEQ ID ID NO: SEQ ID NO: 53) 91) NO: 53) STIF1GRMZM2G016434 1051 + TAAAATA AGG - GTGTTTC AGG + GGACCG AGG ortholog(SEQ ID NO: 13) (SEQ ID AGATGGT GAACGT AAGGA NO: 54) ACAAGA AAACTCGGAGTAA (nt 1002- (SEQ ID ATT (nt 1021 of NO: 92) 267-286 SEQ ID of SEQNO: 54) ID NO: 54) BMY1 GRMZM2G384528 1380 + CTCCGCT CGG + GCGTGTT GGG -CAACGG AGG ortholog (SEQ ID NO: 23) (SEQ ID CTCTCAA GGCAAG CGACGANO: 55) ACTCCC CCCGCTC AACGA (nt 1232- (nt 724-743 GTG 1251 of of SEQ ID(SEQ ID SEQ ID NO: 55) NO: 93) NO: 55) ¹Sequence before the ATG of thecoding sequence (CDS) of the transcription factor includes at least 1000bp of sequence upstream of the 5′UTR predicted by Phytozyme and/ortranscript analysis, which is a variable length for each gene. ²Guides#1, #2, #3 are DNA molecules encoding the guide portion of sgRNA. Theyare fused to DNA encoding the guide RNA scaffold (gRNA Sc)(i.e. See FIG6A) and the resulting transcribed product is a functional sgRNA (FIG.6B) that has all the elements to bind with the complementary targetgenomic DNA that lies adjacent to a PAM sequence (FIG. 6C), and tointeract with the CAS enzyme. The sequences of the Guides #1, 2, and 3are inherently equivalent to the guide target sequence to which thesgRNA base pairs on the genomic DNA for cleavage, and these positions inthe upstream region of the endogenous transcription factor promoter areillustrated in FIG. 7A. The term ″nt″ refers to nucleotides at positionswithin sequences as specified. ³Strand (+/-) refers to the sgRNA bindingto either the forward strand of DNA (+) or its reverse complement (-).⁴PAM refers to the protospacer adjacent motif that resides directlyadjacent to the 3′ end of the guide target site (FIG. 6C).

TABLE 6 Promoter regions for additional maize orthologs to switchgrasstranscription factors STR1, STIF, and BMY1 Length of upstream regionMaize Gene Gene locus name from CDS used for analysis¹ STR1 orthologGRMZM2G018398 1378 (SEQ ID NO: 3) (SEQ ID NO: 75) GRMZM2G171179 1387(SEQ ID NO: 7) (SEQ ID NO: 76) GRMZM2G018984 1565 (SEQ ID NO: 9) (SEQ IDNO: 77) STIF ortholog GRMZM2G087059 1200 (SEQ ID NO: 15) (SEQ ID NO: 78)GRMZM2G425798 1200 (SEQ ID NO: 17) (SEQ ID NO: 79) GRMZM2G309731 1200(SEQ ID NO: 19) (SEQ ID NO: 80) GRMZM2G457562 1538 (SEQ ID NO: 40) (SEQID NO: 81) GRMZM2G100727 1200 (SEQ ID NO: 42) (SEQ ID NO: 82) BMY1ortholog GRMZM2G180947 1689 (SEQ ID NO: 25) (SEQ ID NO: 83)GRMZM2G064426 1480 (SEQ ID NO: 27) (SEQ ID NO: 84) GRMZM5G804893 1476(SEQ ID NO: 29) (SEQ ID NO: 85) GRMZM2G303465 1200 (SEQ ID NO: 47) (SEQID NO: 86) ¹Sequence before the ATG of the coding sequence (CDS) of thetranscription factor includes 1200 bp of sequence upstream of the 5′UTRpredicted by Phytozyme and/or transcript analysis, which is a variablelength for each gene (See FIG. 7).

Design of promoter insertion cassette: The promoter insertion cassettecontains the promoter to be inserted flanked by DNA that is homologousto each side of the CRISPR/Cas nuclease cut site. An illustration of apromoter insertion cassette for promoter X is shown in FIG. 7B. Theflanking DNA fragments direct the promoter cassette insertion into thecut genomic DNA which is subsequently repaired through the plantsendogenous homology directed repair mechanism. In the example in FIG. 7,guide target sites #1 and #3 have been used to excise DNA in thepromoter region upstream of the transcription factor. The flankingregions for the promoter insertion cassette are thus designed to behomologous to DNA upstream of the guide target site #3 nuclease cut siteand downstream of the guide target site #1 nuclease cut site.

The promoter to be inserted can be selected from the large number ofpromoters active in plant cells, including the promoters listed in TABLE3 and TABLE 4. Promoters can be selected based on the desired strengthand intended tissue specific expression pattern for the transcriptionfactor. Liu & Stewart (2016, Current Opinion in Biotechnology, 37, 36)have described synthetic promoters that are active in plants cells andthese can also be used to enable the invention. Based on the disclosureherein, it will be apparent to a person of skill in the art that TABLE 3and TABLE 4 represent examples of promoters that can be used and thatthere are other promoters that are active in plants that can besubstituted for these promoters.

Depending on the method for delivering the promoter insertion cassetteto the plant, it may be advantageous to flank the insertion cassettewith sgRNA binding sequences to release the insertion cassette in thepresence of active Cas9 (FIG. 7B). For example, if the insertioncassette is delivered on a plasmid from transfection of protoplasts orparticle bombardment transformation procedures, flanking the insertioncassettes with sgRNA binding sites and adjacent PAM sequences willrelease the insertion cassette in the presence of Cas9. If the deliverymethod is via Agrobacterium-mediated plant transformation, flanking theinsertion cassettes with the sgRNA binding sites and PAM sequences willrelease the insertion cassette from the T-DNA in the presence of Cas9.This may expedite insertion.

Genetic Constructs for Replacing the Promoter for Expression ofGRMZM2G384528 (SEQ ID NO: 23), a Maize Ortholog of the Switchgrass BMY1Transcription Factor:

Promoter replacement through Agrobacterium-mediated transformation:Binary vector pYTEN30 (FIG. 8A, SEQ ID NO: 71) contains expressioncassettes containing the Guide 1 and 3 DNA fragments in TABLE 5. TheseDNA fragments can each be fused to a DNA fragment encoding a guide RNAscaffold to form functional sgRNAs that together can excise a portion ofthe endogenous promoter of the switchgrass BMY1 maize ortholog encodedby GRMZM2G384528 (SEQ ID NO: 23) (TABLE 1). The Guide 1 and 3 DNAfragments in TABLE 5 encode the guide portion of a sgRNA and weredesigned as described in FIG. 7 using a 1380 bp maize genomic DNAfragment downloaded from Phytozome (SEQ ID NO: 55), encompassing the5′UTR of the GRMZM2G384528 gene plus an additional ˜1 kb DNA upstream ofthe 5′UTR in the promoter region of GRMZM2G384528. In transformationvector pYTEN30, the Guide 1 and 3 DNA are each fused with DNA encodingsgRNA scaffolds and the resulting DNA fragments are expressed inseparate expression cassettes under the control of the rice U6 promoter.Transformation vector pYTEN30 also contains the Cas9 enzyme codonoptimized for rice expressed from a double enhanced CaMV 35S promoter,and the hptI gene (containing a CAT-1 intron) for selection oftransformants with hygromycin expressed from a double enhanced CaMV 35Spromoter fused to an hsp70 intron.

The T0 plants obtained from Agrobacterium transformation are examinedfor CAS9 mediated DNA insertions as follows: During growth, leafmaterial from the T0 transformants is harvested and DNA is extractedfrom the plant tissue using DNA extraction procedures well known in theart. There are multiple commercially available kits, such as the QiagenPlant DNeasy kit, that can be employed for this purpose. PCR reactionsare performed using primers that bind to regions of genomic DNA about100 base pairs away from the guide #1 and #3 target sites (FIG. 7A).Sequencing analysis is performed on the crude PCR mixture using aNext-Generation sequencing technology and automated sequencing assemblyoffered by a vendor. Plants with insertions are identified and allowedto grow in a greenhouse to maturity prior to seed harvest (T1generation).

T1 seeds are planted and grown in a greenhouse, leaf tissue isharvested, and genomic DNA is isolated. Lines are screened for thepresence of the selectable marker gene and/or the Cas9 gene by PCR.Plants that no longer have these genes may have lost the DNA encodingthe Cas9 machinery but may still retain the DNA insertion. Retention ofthe edit in plants that have lost the Cas9 gene is performed using NextGeneration Sequencing. Screening for loss of the Cas9 gene can also bedone by co-expressing a visual marker such as DsRed, a red fluorescentprotein from the Discoma genus of coral (Matz et al., 1999, Nat.Biotechnol. 17, 969-973), by placing an expression cassette coding thegene within the T-DNA region of the vector to allow visual detection ofseeds that no longer carry the vector encoded transgenes. T1 transgenefree plants are thus further screened for edits by extracting genomicDNA from leaf tissue and performing PCR reactions using primers thatbind to regions of genomic DNA about 100 base pairs away from the sgRNAbinding site. Sequencing analysis is performed on the crude PCR mixtureusing a Next-Generation sequencing technology and automated sequencingassembly offered by a vendor. Plants with insertions are identified.Lines with identified insertions that do not contain T-DNA containingthe Cas9 gene are identified and allowed to grow in a greenhouse tomaturity prior to seed harvest (T2 generation). The expression levels ofthe transcription factor in various tissues is determined. Transcriptlevels in seedlings, leaves, stem tissues, roots, silks, cobs, and seedsat different developmental stages are determined by RT-PCR using a genesuch as β-actin as a reference. There are multiple methods forextracting total RNA, including through the use of commerciallyavailable kits, such as the RNeasy Plant Mini Kit from Qiagen (Valencia,Calif., USA). The RNAeasy Plant Mini Kit from Qiagen is used accordingto the manufacturer's protocol. DNase treatment and column purificationare performed and RNA quality is assessed using an Agilent 2100Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA) accordingto the manufacturer's instructions. The RT-PCR analysis is performedwith 50 ng of total RNA using a One Step RT-PCR Kit (Qiagen, Valencia,Calif., USA). Lines with increased expression of the transcriptionfactor are selected and evaluated for yield and stress tolerance asdescribed above.

If required, lines can be grown another generation to obtain homozygousplants with the DNA insertion.

Maize lines are evaluated for their total seed yield and other agronomicparameters such as drought tolerance, stress tolerance, stem thickness,number of cobs, size of cobs. The 100 seed weight of the maize seed canalso be analyzed and high yielding lines or lines with good agronomicparameters indicating improved performance as compared the controlplants are advanced.

Promoter replacement through protoplast transfections: Construct YTEN31(FIG. 8B, SEQ ID NO: 72) is a non-binary vector designed for removal ofthe endogenous promoter from GRMZM2G384528 (SEQ ID NO: 23) andreplacement of the endogenous promoter with the maize ubiquitin promoterand intron (SEQ ID NO: 70). This construct can be transformed intomaize. The production of maize protoplasts and their transformation hasbeen previously described by Rhodes and Gray (Rhodes, C. A. and D. W.Gray, Transformation and regeneration of maize protoplasts, in PlantTissue Culture Manual: Supplement 7, K. Lindsey, Editor. 1997, SpringerNetherlands: Dordrecht. p. 353-365).

Promoter replacement through genetic transformation through biolisticprocedures: Site directed insertion of a DNA fragment into maize embryosthrough homology directed repair using biolistic transformationprocedures has been previously described by Svitashev et al. (2015,Plant Physiol., 169, 931). Construct YTEN31 can be used for promoterreplacement of the GRMZM2G384528 (SEQ ID NO: 23) using the proceduresfor generation of maize embryos, biolistic transformation, andregeneration of plants as described by Svitashev et al. Alternatively,nanotube or nanoparticle mediated DNA delivery can be used (Kwak et al.,2019, Nature Nanotechnology, DOI 10.1038/s41565-019-0375-4) (Demirer etal, 2019, Nature Nanotechnology, DOI 10.1038/s41565-019-0382-5),

Promoter replacement using ribonucleoprotein complexes:Ribonucleoprotein complexes (RNPs) of Cas9, synthesized sgRNAs, andpromoter insertion cassettes, can be delivered to the appropriate planttissue to achieve promoter replacement. In some cases, appropriatetissue will be protoplasts due to the ease of uptake of the RNPs, andthe ability to produce callus cultures from the protoplasts which cansubsequently be regenerated into plants using appropriate tissue culturemethods. Woo et al. (Nature Biotechnology, 2015, 33, 1162-1164) havedescribed the delivery of RNPs to plant protoplasts and subsequentgenome editing. RNPs can also be delivered using methods employing forexample nanotubes.

DNA construct YTEN32 (FIG. 8C, SEQ ID NO: 73) is designed as a promoterinsertion cassette to replace the endogenous promoter of GRMZM2G384528(SEQ ID NO: 23), a maize ortholog of the switchgrass BMY1 transcriptionfactor, with the maize ubiquitin promoter (SEQ ID NO: 70). Formation ofRNPs using the DNA fragment of YTEN32, purified CAS9 enzyme, and twosynthesized sgRNAs to remove a portion of the GRMZM2G384528 promoter canbe used. The two synthesized sgRNAs are produced containing a guide andscaffold. One sgRNA contains the transcribed Guide #3 sequence forGRMZM2G384528 (TABLE 5) fused to a guide RNA scaffold to form afunctional chimeric guide RNA. The other sgRNA contains the transcribedGuide #1 sequence for GRMZM2G384528 (TABLE 5) fused to a guide RNAscaffold to form a functional chimeric single guide RNA (sgRNA). RNPsare formed as described by Woo et al. (Nature Biotechnology, 2015, 33,1162-1164) and delivered to maize protoplasts that are made aspreviously described by Rhodes and Gray (Rhodes, C. A. and D. W. Gray,Transformation and regeneration of maize protoplasts, in Plant TissueCulture Manual: Supplement 7, K. Lindsey, Editor. 1997, SpringerNetherlands: Dordrecht. p. 353-365).

Cell-penetrating peptides can also be used to deliver RNPs into cells.The delivery of macromolecules with cell-penetrating peptides haspreviously been demonstrated in triticale (Chugh et al., 2009, PlantCell Rep., DOI 10.1007/s00299-009-0692-4) and permeabilized wheatimmature embryos (Chugh and Eudes, 2008, FEBS J., 10, 2403) and can beadapted for use in maize.

Example 5

The expression of a transcription factor can be modulated by insertionof various genetic elements. The replacement of the promoter in front ofthe transcription factor is described above. Other methods formodulating promoter activity include insertion of an intron near the 5′end of the transcription factor gene to achieve more stable expression,or insertion of a transcriptional enhancer sequence to modify theactivity of the promoter. Examples of such insertion cassettes and theirinsertion in plant genomic DNA to modify the strength of a promoter areillustrated in FIG. 9.

There are multiple intron sequences that can be used to enable theinvention, including the HSP70 intron (Brown & Santino, 1997, U.S.patent Ser. No. 05/593,874) and the maize ubiquitin 1 intron.

There are multiple enhancer sequences that can be used to enable theinvention that are capable of enhancing the activity of a plantpromoter, including the enhancer element of the 35S promoter (Kay etal., 1987, Science, 1987, 236, 1299).

Example 6. CRISPR Editing with the CpfI Nuclease

In some cases, it may be desirable to use a nuclease with a differentPAM sequence than the Cas9 enzyme to enable insertion of DNA into plantgenomes. The CpfI class of enzymes have a different PAM sequence,depending on their source, allowing cuts at different genomic sequencesthan Cas9, which has a PAM sequence of “NGG”. There are several CpfIenzymes available (Zetsche et al., 2015, Cell, 163, 759; Gao et al.,2017, Nature Biotech., doi:10.1038/nbt.3900; Tang et al., 2017, NatPlants, 3, Article number 17018; Wang et al., Molecular Plant, 2017, 10,1011; Begemann et al., 2017, Scientific Reports, 7, 11606), some whichare listed in TABLE 7 with their corresponding PAM sequences, all ofwhich are useful for practicing this invention.

The CpfI enzyme produces double stranded DNA breaks with nucleotideoverhangs, whereas Cas9 produces blunt ends. Engineering similarnucleotide overhangs on the DNA fragment to be inserted might improveinsertion (Li et al., 2018, Journal of Experimental Botany, 69, 4715).CpfI enzyme also does not need a tracrRNA to be functional, thus sgRNAsfor this enzyme are shorter.

Examples of using Cpf1 enzymes for genome insertion in plants includeBegemann et al. (2017, Scientific Reports, 7, 11606), Li et al. (2018,Journal of Experimental Botany, 69, 4715).

TABLE 7 Cpf1 enzymes and their variants useful for genome editing Cpf1Enzyme Source PAM¹ AsCpf1 Acidaminococcus sp. TTTV BV3L6 AsCpf1S542R/K607R AsCpf1 variant TYCV AsCpf1 S542R/K548V/N552R AsCpf1 variantTATV LbCpf1 Lachnospiraceae TTTV bacterium ND2006 LbCpf1 G532R/K595RLbCpf1 variant TYCV FnCpf1 Francisella novicida TTN U112(NC_008601)¹Abbreviations in PAM consensus sequences; Y = C or T; V = A, C, or G; N= any base

The ability of the CpfI enzyme to cleave its own CRISPR RNA also allowsan array of sgRNAs to be arranged on a single genetic fragment which issubsequently cleaved by CpfI to initiate multiplex editing (Zetsche etal., 2017, Nature Biotech, 35, 31-34).

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The material in the ASCII text file, named“YTEN-58988WO-Sequence-Listing_ST25.txt”, created Apr. 1, 2019, filesize of 233,472 bytes, is hereby incorporated by reference.

1. A method for modifying a corn plant, the method comprisingupregulating, in the corn plant, one or more polynucleotides orpolypeptides selected from among the following: (a) one or morepolypeptides comprising SEQ ID NOS: 87, 88, or 89; (b) one or morepolypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16, 18, 20, 24, 26,28, 30, 32, 41, 43 or 48; (c) one or more of the polypeptides set forthin (a) having at least 85%, 90%, 95% or higher sequence identity to oneor more of the polypeptides set forth in (b); (d) one or morepolynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23,25, 27, 29, 31, 40, 42 or 47; (e) one or more polynucleotides having atleast 85%, 90%, 95% or higher sequence identity to one or more of thepolynucleotides set forth in (d); or (f) one or more polypeptidesencoded by one or more of the polynucleotides set forth in (d) or (e).2. The method of claim 1, further comprising growing the modified plantunder conditions whereby the modified plant exhibits one or moreenhanced characteristics as compared to a control plant grown undersimilar conditions.
 3. The method of claim 1, wherein the one or moreupregulated polynucleotides comprise SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17,19, 23, 25, 27, 29, 31, 40, 42 or
 47. 4. The method of claim 1, whereinthe one or more upregulated polynucleotides or polypeptides exhibit atleast a two-fold change in expression as compared to that of a controlplant.
 5. The method of claim 4, wherein the change in expression isaccomplished by introducing a transgene for one or more globaltranscription factors, wherein the transgene comprises a polynucleotideselected from SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, 29,31, 40, 42 or
 47. 6. The method of claim 1, wherein the one or moreupregulated polynucleotides or polypeptides are upregulated by insertionand/or substitution of one or more nucleotides, site-specificmutagenesis, chemical mutagenesis, targeting induced local lesions ingenomes (TILLING), gene editing techniques using CRISPR nucleaseselected from Cas nuclease, Cas9 nuclease, CasX nuclease, CasY nuclease,a Cpf1 nuclease, a C2c1 nuclease, a C2c2 nuclease (Cas13a nuclease), ora C2c3 nuclease, NgAgo nuclease, TALEN or ZFN techniques.
 7. The methodof claim 6, wherein the one or more upregulated polynucleotides orpolypeptides are upregulated by targeting one or more guidepolynucleotides to one or more target sites selected from a promoter, aterminator, or a coding sequence of the one or more polynucleotides setforth in (d) or (e).
 8. The method of claim 1, wherein the modifiedplant exhibits one or more enhanced characteristics selected from higherphotosynthesis rates, higher photosynthetic electron transport rates,higher non-photochemical quenching, reduced photorespiration rates,higher biomass yield or content, higher seed yield, improved harvestindex, higher seed oil content, improved nutritional composition,improved nitrogen use efficiency, drought resistance, flood resistance,disease resistance, salt tolerance, higher CO₂ assimilation rate, orlower transpiration rate.
 9. The method of claim 8, wherein the modifiedplant exhibits an increase in seed oil content or seed yield as comparedto a control plant.
 10. The method of claim 9, wherein the seed oilcontent of the modified plant is increased by 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100% or higher relative to the control plant. 11.The method of claim 8, wherein the modified plant exhibits an increasein photosynthetic electron transport rate as compared to a controlplant.
 12. The method of claim 11, wherein the photosynthetic electrontransport rate of the modified plant is increased by 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100% or higher relative to the controlplant.
 13. A modified corn plant comprising: (a) one or morepolypeptides comprising SEQ ID NOS: 87, 88, or 89; (b) one or morepolypeptides comprising SEQ ID NOS: 4, 6, 8, 10, 14, 16, 18, 20, 24, 26,28, 30, 32, 41, 43 or 48; (c) one or more of the polypeptides set forthin (a) having at least 85%, 90%, 95% or higher sequence identity to oneor more of the polypeptides set forth in (b); (d) one or morepolynucleotides comprising SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23,25, 27, 29, 31, 40, 42 or 47; (e) one or more polynucleotides having atleast 85%, 90%, 95% or higher sequence identity to one or more of thepolynucleotides set forth in (d); or (f) one or more polypeptidesencoded by one or more of the polynucleotides set forth in (d); whereinthe one or more polypeptides of (a), (b), (c), or (f) or the one or morepolynucleotides of (d) or (e) are upregulated.
 14. The modified plant ofclaim 13, wherein the modified plant exhibits one or more enhancedcharacteristics as compared to a control plant grown under similarconditions.
 15. The modified plant of claim 13, wherein the one or moreupregulated polynucleotides or polypeptides exhibit at least a two-foldchange in expression as compared to that of a control plant.
 16. Themodified plant of claim 15, wherein the change in expression isaccomplished by introducing a transgene for one or more globaltranscription factors, wherein the transgene comprises a polynucleotideselected from SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, 19, 23, 25, 27, 29,31, 40, 42 or
 47. 17. The modified plant of claim 13, wherein the one ormore upregulated polynucleotides or polypeptides are upregulated byinsertion and/or substitution of one or more nucleotides, site-specificmutagenesis, chemical mutagenesis, targeting induced local lesions ingenomes (TILLING), gene editing techniques using CRISPR nucleaseselected from Cas nuclease, Cas9 nuclease, CasX nuclease, CasY nuclease,a Cpf1 nuclease, a C2c1 nuclease, a C2c2 nuclease (Cas13a nuclease), ora C2c3 nuclease, NgAgo nuclease, TALEN or ZFN techniques
 18. Themodified plant of claim 17, wherein the one or more upregulatedpolynucleotides or polypeptides are upregulated by targeting one or moreguide polynucleotides to one or more target sites selected from apromoter, a terminator, or a coding sequence of the one or morepolynucleotides set forth in (d) or (e).
 19. The modified plant of claim13, wherein the modified plant comprises one or more enhancedcharacteristics selected from higher photosynthesis rates, higherphotosynthetic electron transport rate, higher non-photochemicalquenching, reduced photorespiration rates, higher biomass yield orcontent, higher seed yield, improved harvest index, higher seed oilcontent, improved nutritional composition, improved nitrogen useefficiency, drought resistance, flood resistance, disease resistance,salt tolerance, higher CO₂ assimilation rate, or lower transpirationrate.
 20. The modified plant of claim 19, wherein the modified plantexhibits an increase in seed oil content or seed yield as compared to acontrol plant. 21-25. (canceled)